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Transcription Attenuation
ROBERT LANDICK, CHARLES L. TURNBOUGH, JR., AND
CHARLES YANOFSKY
81
Introduction / 1263
Other Interesting Examples / 1280
Transcription Attenuation Mechanisms in Other
Organisms / 1281
B. subtilis trp Operon / 1282
B. subtilis pyr Operon / 1282
tRNA-Directed Transcription
Antitermination / 1282
Possibility of Transcription Attenuation in
Eukaryotes / 1282
Early HistOlY / 1264
Objectives of Transcription Attenuation as a
Regulatory Mechanism / 1264
Attenuation Mechanisms in Enteric
Bacteria / 1264
Class I: Ribosome Stalling, Alternative RNA
Structure Formation / 1264
Class II: Ribosome-RNA Polymerase
Coupling / 1273
Class III: Regulatory Factor Dependent
1278
Class IV: Rho Factor Dependent / 1278
Evolutionary Aspects, Conclusions, and
Expectations / 1282
LITERATURE CITED / 1283
It was once thought that repression of transcription initiation was sufficiently adaptable as a regulatory mechanism to be
principally responsible for most gene regulation in most organisms. It is now apparent that each organism uses a variety of
regulatory strategies in modulating gene expression. Often a
single gene or operon is regulated by multiple independent
mechanisms. In view of the extensive use of gene regulation, an
important consideration for each organism during the course of
evolution must have been how much genetic information to
devote to regulatory processes. In some organisms, notably bacteria, the need for effective gene regulation must have conflicted
with the desire to maintain a relatively small genome. A small
genome would limit the number of regulatory genes and events
that could be devoted to control and probably would favor regulatory mechanisms that require minimal genetic information.
These evolutionary pressures notwithstanding, it is evident that
even organisms with small genomes, such as viruses, make extensive use of gene regulation.
Once organisms were committed to differential gene regulation, it would have been advantageous to sense all external and
internal events relevant to each gene's expression and to couple
these events in one or more regulatory circuits. The use of both
gene-specific regulatory mechanisms and global regulatory
mechanisms that interrelated gene activities clearly would have
been beneficial. It is perhaps not surprising that so many regulatory processes exist and that cells can sense and respond in so
many ways to the numerous events that influence their behavior.
In this chapter, we focus on transcription attenuation in Escherichia coli and Salmonella typhimurium (official designation,
Salmonella enterica serovar Typhimurium). We shall review the
INTRODUCTION
As many of the features of gene expression and its regulation have
been revealed, it has become apparent that every organism, from
the simplest to the most complex, utilizes an appreciable fraction
of its resources in regulatory processes that control its functional
genes and interrelate their various activities. The development of
regulatory mechanisms optimal for each organism, in its environmental niche, is thus a major theme in evolution. Macromolecular interactions are the principal events of gene regulation, with small and large molecules serving as signals for these
regulatory activities.
Most prokaryotic operons are regulated transcriptionally at
or near a short promoter region that binds relatively few regulatory proteins. These interactions may activate or inhibit transcription initiation. Regulatory mechanisms also target
molecular events that occur subsequent to transcription initiation. These mechanisms include processes that influence tran.script elongation and transcription termination. These are
classified under the general heading of transcription attenuation, the subject ofthis article. In most eukaryotes, regulation'
of gene expression is more complex, with each gene's promoter/regulatory region replete with regulatory sites, or elements, that allow recognition of, and response to, many different
regulatory proteins. The state of these proteins reflects their
identity, the cell's environment, the stage of development, and
the metabolic and other activities that are proceeding in the cells
in which these regulatory proteins reside. Transcription attenuation also occurs in eukaryotes, but features of such mechanisms
are only beginning to emerge.
1263
1264
LANDICK ET AL.
features of the best-understood attenuation mechanisms that
are used by these organisms. Transcription attenuation was reviewed in the previous edition of this volumtt (91) and, most
recently, in the Cold Spring Harbor Laboratory monograph
Transcriptional Regulation (89).
Early History
During the 1950s, there was considerable progress in our understanding of transcriptional regulation in bacteria, largely as a
result of studies by Jacob and Monod on negative control (inhibition of transcription initiation) of lac operon expression in E.
coli (65). Subsequent studies with the arabinose operon of E. coli
established that positive control (activation of transcription initiation) also was used to regulate gene expression (41). During
this period, several investigations with mutant tRNA synthetases
suggested that these enzymes, or some consequence of their
activity, affected expression of amino acid biosynthetic operons
(39, 144). Extensive mutational studies on the mechanism of
transcriptional regulation of the histidine (his) biosynthetic operon of S. typhimurium implicated histidinyl-tRNA synthetase,
the charging and function of tRNAHis, and/or the utilization of
histidine in translation of the early segment of the his operon
transcript as the molecules and events that modulated his operon
expression (9). These studies failed to detect a repressor gene or
protein, suggesting that some other regulatory mechanism was
employed (9). During this period, regulatory studies with the trp
operon of E. coli mostly dealt with the role of the trp repressor in
negative regulation of transcription initiation in this operon.
Several observations made during the course of these studies
indicated that a tryptophan -specific regulatory mechanism other
than repression must influence this operon's expression. Thus,
kinetic studies of transcription within the initial segment of the
tIP operon (the leader region) indicated that the addition of
tryptophan to a tryptophan-starved culture caused premature
termination of transcription within this initially transcribed region of the operon (63). It was also observed that mutant strains
that lacked a functional ttP repressor responded to tryptophan
starvation by increasing trp operon transcription (12). In addition, some tryptophanyl-tRNA synthetase mutants were found
to have elevated levels of tIP operon enzymes even when grown
in the presence of excess tryptophan (116).
Subsequent studies with both the his operon of S. typhimurium and the trp operon of E. coli demonstrated that internal
deletions that removed portions of the leader region but left the
promoter intact caused a large increase in synthesis of operon
mRNA and proteins without affecting mRNA stability (17, 64).
These findings suggested that a natural barrier to transcription
elongation, such as a transcription termination site, was located
in the leader regions of these operons. This barrier was presumably removed in the deletion mutants (17, 64, 78). Kasai (78)
introduced the use of the term "attenuation" to describe modulation of his operon expression at such a barrier. The barrier is
now Imown to be a transcription termination site, referred to as
an attenuator (78). Transcription attenuation mechanisms canj
be separated into distinct classes, many of which will be considered here. The term "translation attenuation" has been used to
describe processes with somewhat similar features in which gene
expression is regulated by events that influence the ability of a
ribosome to initiate translation. In this chapter we shall describe
only transcription attenuation mechanisms.
Objectives of Transcription Attenuation as a
Regulatory Mechanism
Transcription attenuation allows an organism to regulate gene
expression by exploiting RNA sequences and structures, as opposed to information contained in DNA. It also allows cells to
sense availability of the precursors needed for RNA and protein
synthesis. An RNA signal can direct a transcribing RNA polymerase molecule to pause during transcript elongation, to terminate transcription prematurely, or to transcribe through a potential termination sequence. RNA sequences also can provide
binding sites for regulatory factors that can influence the aforementioned events. Mutually exclusive RNA structures can allow
determination of whether transcription will 01' will not be terminated at a particular site. By exploiting RNA sequences and
structures in regulatory decisions, nature has engaged RNAs in
ways that are not appropriate to regulation of transcription
initiation. Thus, we see that translation is often used to mediate
attenuation decisions. Similarly, a transcribing or paused RNA
polymerase molecule itself, its associated transcription factors, 01'
the Rho termination factor can serve as a regulatory target,
thereby influencing attenuation. Transcription attenuation
mechanisms therefore increase the repertoire of molecular events
that an organism can exploit in optimizing gene expression.
A second attractive feature of some transcription attenuation
mechanisms, as we shall see from examples described in this
chapter, is that relatively little genetic information, often less
than 100 nucleotides (nt), is needed for specific regulation. Also,
in many instances transcription attenuation proceeds without
the participation of a specific regulatory protein. It seems likely,
therefore, that if a primordial RNA world preceded the DNA
world we Imow today, transcription attenuation mechanisms
probably would have been among the earliest regulatory mechanisms to have evolved.
ATTENUATION MECHANISMS IN ENTERIC BACTERIA
The hallmark of transcription attenuation is control over the
continuation of transcript elongation at sites that are encountered by RNA polymerase prior to a particular gene. A wide
variety of attenuation mechanisms have been discovered in enteric bacteria, and many examples fall into classes that share
certain features (89). The four principal classes of attenuation
mechanisms (Table 1) are (i) a class in which the location of a
ribosome controls formation of alternative secondary structures
in the nascent transcript (ribosome stalling, alternative RNA
structure-dependent attenuation); (ii) a class in which coupling
between ribosome and RNA polymerase movement can directly
preclude formation of a terminator RNA hairpin (ribosome
coupling-dependent attenuation); (iii) a class in which a transacting factor governs formation of a terminator structure by
interacting with the nascent transcript (regulatory factor-dependent attenuation); and (iv) a class in which the action of the
Rho termination protein in the RNA segment preceding a structural gene is regulated (Rho-dependent attenuation). We will
consider these classes individually in the fonowing sections.
Ci::lass I: Ribosome Stalling, Alternative RNA
Structure Formation
As described above, transcription attenuation was discovered in
regulatory studies with the his and trp amino acid biosynthetic
I.
[
'I·
!
~!
j
81
TRANSCRIPTION ATTENUATION
1265
TABLE 1 Mechanisms of transcription attenuation in bacteria
Class"
Operon
III
Amino acid biosynthesis,
tRNA synthetase
pyrBl, pyrE (pyrimidine
biosynthesis)
ampC (p-Iactamase)
Amino acid biosynthesis,
tRNA synthetase
bgl (P-glucoside catabolism)
III
III
IV
rpsl (ribosomal protein)
trp, pyr (biosynthesis)
tna (Trp catabolism)
II
II
III
Species
Sigilal
Mediator
Event regulated
Many
Uncharged tRNA
Ribosome
RNA secondary structure
Enterics
UTP deficiency
Ribosome
RNA secondary structure
Some enterics
Bacilli, gram-positive bacteria
Ribosome content
Uncharged tRNA
Ribosome
tRNA
RNA secondary structure
RNA secondary structure
E. coli and others
p-Glu~oside phosphorylation
RNA secondary structure
E. coli
rRNAlevel
Trp or Pyr level
Extracellular Trp
Regulatory protein
phosphorylation
L4protein
Regulatory protein
Leader peptide?
Bacilli, gram-positive bacteria
Enterics
RNA secondary structure?
RNA secondary structure
Rho access
"Class I, mechanisms in which the location of a ribosome controls formation of alternative secondary structures in the nascent transcript (ribosome stalling,
alternative RNA structure-dependent attenuation); class II, mechanisms in which coupling between ribosome and RNA polymerase movement can directly
preclude formation of a terminator RNA hairpin (ribosome coupling-dependent attenuation); class III, mechanisms in which a trans-acting factor governs
formation of a terminator structure by interacting with the nascent transcript (regulatory factor-dependent attenuation); class IV, mechanisms in which the
action of the Rho factor in the RNA segment preceding a structural gene is regulated (Rho-dependent attenuation).
bThe IpS] operon in E. coli encodes Ips]-rpICDWB-IpsS-IpIV-IpsC-IpIP-lpmC-lpsQ. rplD enclodes ribosomal protein L4.
operons in E. coli and S. typhimurium. Subsequent investigations
revealed that closely related mechanisms control termination in
the leader regions ofthe leu (48,81,169), thr (44,107), pheA (62,
186), ilvGMEDA (93, 117), and ilvBN (43,56) amino acid biosynthetic operons, as well as in the leader region of the pheST
operon, which encodes the small (pluS) and large (pheT) subunits of phenylalanyl-tRNA synthetase (153, 154; chapter 91).
The evidence underlying current knowledge of these attenuation mechanisms has been the subject of two recent reviews,
which include a case-by-case account of the published information about each (91), the complete secondary structures of the
alternative RNA structures that can form fi'om each operon's
leader transcript (91), and an up-to-date and detailed account of
the different intermediates and events involved in tiP operon
attenuation (89). Therefore, in what follows we will offer a summary description of the key features of the class I attenuation
mechanisms for amino acid biosynthetic operons and restrict
our attention primarily to recent findings and to the key questions that remain unanswered. The reader should refer to recent
reviews (55, 89, 91) for additional details.
Common Features of Class I Attenuation Mechanisms. The structural features of the leader regions of amino acid biosynthetic
operons regulated by class I attenuation mechanisms are surprisingly similar. These similar features include sites of transcription pausing and Rho-independent termination, a coding
region for a short leader peptide containing co dons for the
regulating amino acid(s), and transcript segments that specify
alternative RNA secondary structures (Fig. 1). Transcription
pausing occurs in the initial segment of the leader region and is
caused, in part, by formation of an RNA secondary structure in
the nascent RNA chain, termed the pause RNA hairpin. The
temporary halt to transcription. induced by the pause signal
allows time for a ribosome to begin synthesis of the leader
peptide before the Rho-independent termination site is transcribed. Resumption of transcription when the ribosome encounters the paused polymerase is largely responsible for the
synchronization of transcription and translation that is essential to this class of attenuation mechanisms.
The terminator RNA hairpin (3:4) is transcribed fi'om a
segment of DNA that is downstream from the pause signal and
is termed the attenuator. An alternative RNA secondary structure, termed the antiterminator, can form from the downstream segment of the 1:2 (pause) structure and the upstream
segment of the 3:4 terminator structure. Thus, the antiterminator is designated structure 2:3. Formation of the 2:3 structure
during transcription of the leader region precludes formation
of the terminator, thereby permitting transcription readthrough. The peptide coding region overlaps the promoterproximal segment that is part of the 1:2 RNA structure.
Translation of the peptide coding region versus ribosome stalling at key sites within it selects between terminator and antiterminator formation, causing either transcription termination or
readthrough into the structural genes of the respective operon.
A Detailed Model of Attenuation. To illustrate the role of and
relationship among these different segments of the leader transcript, we will describe our current understanding of events that
occur during transcription of the tiP operon leader region. Here,
a 141-nt leader RNA transcript forms the alternative terminator
(3:4) or antiterminator (2:3) secondary structures (Fig. 2). The
upper portion of the 1:2 structure forms the hairpin portion of
the signal that causes polymerase to pause prior to the 2:3 versus
3:4 decision. In some situations, the entire 1:2 structure forms
after polymerase escapes the pause (see below). The leader RNA
also encodes a 14-amino-acid peptide that contains two tryptophan residues, at positions 10 and 11 (Fig. 2). Once RNA polymerase initiates synthesis of an RNA chain and clears the promoter region, it moves rapidly to a pause site located just after
RNA segment 2 (Fig. 2 and 3). This pause is very short-lived in
vivo, probably less than 1 s, but still long relative to the average
dwell time of RNA polymerase at a single DNA position. The
pause signal is complex, consisting of multiple components, one
of which is a nascent transcript RNA hairpin that corresponds to
the upper portion Of the 1:2 secondary structure (discussed below). Halting RNA polymerase at the pause site allows time for
initiation of leader peptide synthesis before RNA polymerase
moves into the attenuator region where the regulatory decision
1266
LANDICK ET AL.
Antitermlnator
Structure
Readthrough
Conformation
AU
Pause
Structure
Alternative RNA
Secondary Structures of
the Leader Transcript
Terminator
Hairpin Structure
}
pause
Hairpin
Termination
Conformation
RNA
DNA
Promoter
Transcription
Start
r
_ _3_ _ _ _L -
2
ATG· • • •
t- _______________ c=
·TGA
Leader Peptide
Coding Region
First
Structural
Gene
Pause Site
Attenuator
FIGURE 1 Structure and relevant features of the leader transcripts and leader regions of amino acid
biosynthetic operons regulated by attenuation. The antiterminator and pause structures are depicted
with unpaired bulges to emphasize that they do not contain perfectly paired stems. Note that the
pause RNA hairpin corresponds to the upper portion of the 1:2 structure and is the only portion of
the structure thought to form when RNA polymerase resides at the pause site. The term "terminator"
describes an RNA or DNA segment that is responsible for, or contributes to, a transcription termination event. Within the context of attenuation, "terminator" refers to the terminator RNA hairpin (here
3:4) plus the run of U's that can form when formation of a mutually exclusive antiterminator
structure (here 2:3) is prevented. (This figure and Fig. 2, 3, 7, 8, and 9 are adapted from reference 89
with permission of the publisher.)
A Termination Conformation
AAGUUCAC
10
1:2
B
A AA
Stop 70 G
C
U
G
S
C=G
Ser uC=Gc
A
A
A
A
Thr
U A A
G
A
C=G1OO
G=C
AU=AA
90C
U
Pause
Hairpin
3:4
U=A
C=G 80
A=U
A
C=G
A
U
G
A{gC
UA
Uc
G
G
UU
1~og~g
G
Trp G=C
C=G 130
U
U=A
'-- C=G
20 A
Trp G=C
C=G
U
Met Lys Ala lie Phe Val Leu Lys Gly G=C90
G=C
CGACAAUGAAAGCAAUUUUCGUACUGAAAGGUU=A
AUCAGAUACCCA=UUUUUUUU
30
40
50
U=A
110
140
/G=C
paUSE GC ~1oo
~
2:3
Antitermination
Conformation
~
C
C
AC=G A
U=A
uA=u
U=A110
uG=cc
BOG
C
AC=G A
G=C
G=C
Trp Trp Arg Thr Ser Stop G=C
"'UGGUGGCGCACUUCCUGAAAC=GCCUAA'"
60
70
120
=
UA A
FIGURE 2 Alternative RNA secondary structures of the tiP operon leader transcript. (A) Termination conformation; (B) antitermination conformation. The positions in the leader peptide corresponding to the initiation, control, and termination codons are shown in boldface.
81
TRANSCRIPTION ATTENUATION
1267
INITIAL STAGES OF TRANSCRIPTION
DNA
-----'-O"'-b.1~~
+--RNA POLYMERASE
LEADER TRANSCRIPT~.
Ribosome
Fails to Bind
Transcript
Transcription Complex
Pauses After 1:2 Synthesis
PPP
)
.1:2
AUG
~
SUPER-
--~
Ribosome Binds) I
to Transcript
~
1:2
RIBOSOME
ATTENUATION
"\
Ribosome Movement
Releases the Paused
Transcription Complex
TRP STARVATION
ADEQUATE TBP
Ribosome Stalls on Trp Codons,
Allowing Formation of 2:3
Ribosome Moves
to Stop Codon
Ribosome
Releases
atStop~
2:3 Formatioi
Prevents 3:4
Formation
I
Transcript
Released
2:3 Forms;
Terminator (3:4)
Blocked
J.
READTHROUGH
BASAL LEVEL
READTHROUGH
1:2 Forms;'
Terminator (3:4)
~ 3~
/V;
~
~
\~
~RMINATION W
Forms
FIGURE 3 A detailed model of transcription attenuation in the trp operon. The nontranscribed
DNA strand is depicted as being extruded to the outside of RNA polymerase and reannealing at the
upstream edge of the transcription complex to be consistent with chemical and nuclease protection
data (see discussion in reference 94). (See text for further description.)
will be made. Once a ribosome reaches the paused polymerase in .
the course of translation, the paused polymerase is released and
resumes RNA synthesis (88).
This synchronization of transcription with ribosome movement over the leader peptide control co dons is a critical feature
of attenuation. Since extreme depletion of the cognate charged
aminoacyl-tRNA leads to essentially complete readthrough at
the attenuator, synchronization of ribosome and RNA polymerase action must be efficient. However, if translation of the
leader peptide coding region fails, for instance when the leader
peptide initiation codon is mutated, RNA polymerase will even-
tually escape from the pause site spontaneously and terminate at
the attenuator with 95% efficiency (178). When amino acids are
not growth limiting and translation proceeds to the stop codon
normally, termination is only 85% efficient (178).
Once RNA polymerase leaves the pause site, five outcomes
are possible (Fig. 3). Which of these actually is achieved is determined by the relative rates of transcription of leader DNA by
RNA polymerase and ribosome movement on the newly synthesized leader RNA chain. If a ribosome does not load onto the
transcript before polymerase spontaneously escapes the pause
site (the situation just described), the transcript subsequently
1268
LANDICKET AL.
folds into the 1:2-3:4 termination conformation efficiently, and
transcription of the operon stops at the attenuator (outcome 1,
superattenuation; Fig. 3). If the ribosome releases the paused
transcription complex (the normal situation), the translating
ribosome typically will halt synthesis at one of two positions,
depending on the availability of charged tryptophanyl-tRNA:
the control Trp codons when the cell is starved for Trp (position
1) or the UGA stop codon when the supply of Trp is adequate
(position 2) (Fig. 2 and 3). If the supply of charged tryptophanyl-tRNA is inadequate (Trp starvation; left side of Fig. 3),
the ribosome halted at the Trp control codons should block
about 13 nt on either side (129). In this situation, formation of
the 1:2 secondary structure is not possible, and the RNA folds
into the 2:3 antitermination conformation. This precludes subsequent formation of the 3:4 terminator structure and leads to
transcription through the attenuator region into the operon's
structural genes (outcome 2, readthrough; Fig. 3). If the supply
of charged tryptophanyl-tRNA is adequate, the translating ribosome will reach the leader peptide stop codon (adequate Trp;
right side of Fig. 3); either it will remain there until after polymerase transcribes the attenuator or it will release before polymerase reaches the attenuator. If the ribosome remain~ at the stop
codon while RNA segment 4 is synthesized, it will block formation of the 2:3 structure. This, in turn, will promote formation
of the 3:4 terminator hairpin, leading to termination of transcription at the attenuator (outcome 3, termination; Fig. 3). If
instead the translating ribosome releases at the stop codon before synthesis of RNA segment 4 is completed, two outcomes are
possible, depending on whether the nascent transcript spontaneously folds into the 1:2 or 2:3 conformation. If 2:3 forms,
readthrough into the operon's structural genes will occur as
described above for Trp starvation (outcome 4, basal-level
readthrough; Fig. 3). If 1:2 forms, termination will ensue (outcome 5; Fig. 3). We estimate that formation of 1:2 or 2:3 occurs
with approximately equal probability (see below).
In the following sections, we will describe our current understanding of the major features of this model, note several potential variations on the above-described mechanism that may
occur in different amino acid biosynthetic operons, and identify
key questions that require further study.
Alternative RNA Structures Modulate Termination. Although
all eight operons that are regulated by this class of attenuation
mechanism encode potentially alternative RNA structures in
their leader RNAs that are consistent with the model described
above, there are significant variations for each in the exact foldingpatterns of these structures (see reference 91). The his operon
leader, for instance, folds into a termination conformation with
three significant stem-loop structures, termed A:B, C:D, and E:F
(the terminator structure), as shown by the workers who first
studied the his attenuator (Fig. 4). The antitermination conformation of this leader contains two significant stem-loop structures, B:C and D:E (Fig. 4). The his leader RNA encodes seven
tandem histidines overlapping segment A, in contrast to the two
Trp co dons found in the trp leader. This probably reflects the
greater sensitivity of the his attenuator to amino acid starvation
(see below). Despite these differences, however, the alternative
pattern of folding still allows a ribosome stalled on RNA segment
A to prevent formation of the terminator hairpin (E:F) because
elimination of the A:B structure allows the B:C structure to form.
B:C prevents formation ofthe C:D structure, which in turn allows
formation of the D:E structure, which then precludes formation
ofE:F.
Another variation occurs in the ilvGMEDA operon, which
encodes enzymes for synthesis of leucine, isoleucine, and valine.
The ilvGMEDA leader RNA structure contains a large bifurcation in the 1:2 RNA structure (Fig. 5). This effectively divides
segment 1 into two portions, termed lA and IB by Hatfield and
coworkers (see reference 55). Here a significant question has
arisen as to whether a ribosome stalled at the tandem isoleucine
ccodons in the loop of the 1:2 structure could prevent base pairing between segments lA and 2, since it would be more than 13
nt from the RNA segments that pair (Fig. 5). Thus, if 1A:2
re-formed when a ribosome halted in response to starvation for
isoleucine, but not starvation for valine or leucine, 2:3 formation could be inhibited and termination might still result. This
scenario led Hatfield to propose a "double-ribosome" model of
attenuation in the ilvGMEDA leader (55). In this model, a second ribosome loads onto the transcript before the attenuation
decision is made and could stop immediately behind one stalled
at the isoleucine codons. This second ribosome would prevent
pairing between segments lA:2.
Despite these variations, the importance of competition between the formation of mutually exclusive RNA secondary structures in controlling attenuation is established beyond reasonable
doubt. Four independent lines of evidence support this conclusion.
First, for the E. coli tIp, S. typhil11 uriul11 his, and E. coli thr leader
regions, many leader RNA base substitutions have been observed to
alter termination (see reference 91 for a review). Outside the leader
peptide coding regions, the effects of all of these substitutions can
be explained by their effects on the relative stabilities of the different
RNA structures. For example, replacement of G-93 with A in the tIP
leader (Fig. 2) or of U-S6 or of C-llS with A in the his leader
(Fig. 4) increases termination at the corresponding attenuator because these substitutions weaken base pairing in the antiterminator
conformation of the leader RNA. Further, the effects of the CllS~A substitution in the his leader are reversed by a compensatory G-157~U substitution on the opposite side of the D:E RNA
secondary structure because base pairing is restored by the second
substitution.
Second, sequential deletions of RNA segments 1,2,3, and 4 have
been examined for both the E. coli (92) and Serratia l11arcesens
(160-162) tIP operons. Again, their effects are consistent with the
model of attenuation outlined above. Deletion of segment 1 greatly
increases readthrough at the attenuator, presumably because the 2:3
structure forms unhindered. Deletions that extend into segment 2
restore termination to high levels; and further deletion into segment 3 or 4 eliminates termination.
Third, nuclease sensitivity studies with the isolated tIP leader
RNAs from S. l11arcesens (86) and E.(coli (128) reveal patterns of
cleavage and protection by single- and double-strand-specific nucleases that are consistent with the structures depicted in Fig. 2.
Moreover, a recent nuclear magnetic resonance study of a synthetic
RNA corresponding to residues + 114 to +134 of the tIP leader RNA
is consistent with the assigned structure (134).
Finally, the role of the 3:4 terminator hairpin has been studied in
particular detaiL Substitutions of bases in thy paired stems of the
terminator hairpins from the his, tIp, leu, and thr leader regions
have been shown to reduce termination (see references 89 and 91
and references therein). Experiments with both the tIP (143) and
thr (174) leader regions have shown that these substitutions affect
81
A
Termination
Conformation
B
TRANSCRIPTION ATTENUATION
D:E
Antitermination
Conformation
AC
A
E:F
A:B
SIO:a~ G U .~ }
~ 160C ~
C
'u
Asp ~~~
Pause
Hairpin
G=C
A=U
U=A
~~g
Pro C=G90
G=C
U C=G
C=G
C
His AC=G
C=G
70U=A
150C=G170
~Phe
His A=U
l CG g~g
50 A
C=Gu
G=C G=C
A Lys
C=G
U=A A=U
A
60 His A=U)oo
A=U A=U
180
CACCACCAUCAUC=GC =GUGGUGC =GAGA = UUUUUUU
His His His His
U=A
130
/'G=C
PRUSE
~;;;3 120
A=U
G=C
A=U
... ~JU8~tcWcci~Lc
40
A Gin
A
U
C
110 A
1269
B:C
G
G=C
U=A
A=U140
130 C=GA G
G
A
G
U
A
AU U
G=cGA
G
G
U
G=C
C=G1oo
U
C 150
G=C
G=C
90G
A
C
A=U
C=G
C=G
C=G
U
G 120U=A
U=A
U=A
U=A
C=G
C=G
U=A
70
80 U=A
A=U
••• CACCAUCAUCCUGACUAG =CAUUCAG =CAUCUUCCGG •••
His His His Pro AspSIOp 110
160
A
G
Uu CA
C:D
FIGURE 4 Alternative RNA secondary structures of the his operon leader transcript. (A) Termination conformation; (B) antitermination conformation.
transcription only when present in the transcribed DNA strand,
and thus the RNA transcript, and have no effect if present only
in the nontranscribed strand of a heteroduplex template.
Role of the Leader Peptide Coding Region. In the attenuation mechanisms that control expression of amino acid biosynthetic operons,
regulatory information is transduced through synthesis of the
leader peptide. In essence, the rate of ribosome movement over the
control codons senses the availability of cognate charged tRNAs for
A
protein synthesis and communicates this information to the
transcription complex via its influence on nascent RNA structure. Thus, the leader peptide coding region plays a central role
in these mechanisms. Many aspects of this role have been covered in detail in earlier reviews (89,91).Weemphasizefourcentral
points here.
First, translational capacity is sensed through the availability of
charged tRNAs for efficient incorporation into the leader peptide,
not by the actual pools of amino acids. This is shown most conclu-
2:3
1:2
Termination
Conformation
Antitermination
Conformation
A U lie
U
U
lie U=A
A=U lie
G=C
Val UG=C
80G=C Pro
Vel U=A
G=C
G=C Pro
Val U=G
G=U
Ser C=G 100 Cys
LeuVel lie U ~=C
60
Vel U
\
CC U GG
AG
Ser G
U CG U
A
II 11111111
G
1B
AUUCCUUCG
~
C
Uu
lie
ud'Gg~
S
Leu U
C
A A
G
A
C=G
~;;;S
/
1A
PRUSE
Slop Au=f
U=A
Ala C=G
G=C140
G=C
Lys A=U
GIYri'A=UA
A
120AG
C
Arg
G
A
Gly G
A
f
A ~ Leu U
C
C
~
Vel rg Leu UC=G G/y
160 g;:;;g
A
C=G
C=G
U
Ala 40C=G
C=G
G
Thr G=C Ala
C=G
C
Mel A=UAlaLeu Gly Arg Gly
C=G
2~GAAAAGACAAAAUGAC=GCACUUGGA?G~~GA~~~AACGAACUAAGA~~~UUUUUU
A
GAGA
A
Ala
C~
C
U
A
A
AG
Ala CG=CC
G=C
Gly ~~8160
C=G
Cys 1oo~;;X
Pro G=C
Val Vel Val lie lie /Ie Pro C C
••• GUGGUGGUGAUUAUUAUCCCAC = GAAAGG···
80
Lys g;:;;g140
Ala C=G
U=A
U=A
Slop A
C
G
U
AGA
FIGURE 5 Alternative RNA secondary structures of the ilvGMEDA operon leader transcript. (A)
Termination conformation; (B) antitermination conformation.
1270
LANDICK ET AL.
rare codon is sufficient to confer regulation (61), whereas the
phe and his operons each contain seven co dons for a single
regulatory amino acid. Presumably this reflects both the relative
ease of translating a particular codon and the degree of sensitivity to amino acid starvation desired for a particular operon.
Although all of the leader peptide coding regions begin between
nt 22 and 45 of the different leader transcripts, some extend only
to the loop region of the 1:2 hairpin, whereas others continue
over segment 2 to near or past the pause site. Interestingly, the
. aromatic amino acid biosynthetic operons all fall into the first
class and the branched-chain amino acid biosynthetic operons
fall into the second. Presumably this reflects an early divergence
in the evolutionary paths of amino acid biosynthetic operons. It
also may have regulatory significance. In the double-ribosome
model of ilvGMEDA operon attenuation, pausing conceivably
could be induced by base pairing between segments 1A and 2 even
after a ribosome has passed segment 1A (55). Continuation of the
leader peptide coding region up to or past the pause site would
allow the ribosome to release such a paused RNA polymerase.
Fourth, the range of regulation conferred by the different
leader peptide coding regions varies dramatically among the
different operons. The presence or absence of Trp in the growth
medium does not normally affect readthrough of the trp attenuator; the la-fold change in readthrough occurs only upon extreme Trp starvation in mutant bacteria or transiently upon
transfer of bacteria from a Trp-containing to a Trp-free medium
(177). Thus, the trp attenuator may represent adaptation of the
mechanism to an operon also regulated by repression (see discussion in reference 89). In other cases, significant changes in
sively from the findings that defects in the tRNA that reduce its
translational efficiency (e.g., failure to isopentenylate tRNATrp in
a miaA strain [179] or to pseudouridylate tRNAHis in a hisT
strain [72, 99]) or defects in the aminoacyl-tRNA synthetases
responsible for charging tRNATrp or tRNAHis (99, 179) lead to
elevated readthrough at the corresponding attenuators.
Second, it is the act of translation and not the leader peptide
itself that regulates attenuation. Synthesis of the complete thl'
leader peptide was observed in coupled transcription-translation experiments (108), and the tIP leader peptide has been
detected both in vitro (32) and in vivo (35). However, all tests for
trans action have been negative. Further, the leader peptides
themselves are unstable, having half-lives of less than 5 min in
cell extracts (32, 108). At least for the tIP operon, sequences
present downstream from the attenuator site fold back and pair
with the leader peptide ribosome-binding site, effectively precluding more than a single round of leader peptide synthesis
(32). Finally, where a single amino acid is encoded by both rare
and frequently used co dons, such as in the leu operon, the presence of rare codons in the leader peptide coding region confers a
strong regulatory response, whereas the presence of common
co dons does not (14; reviewed in reference 89).
Third, the number and positions of control co'dons within
the leader peptide coding regions vary among the different operons regulated by attenuation (Fig. 6). Some leader peptide
coding regions (tip, his, phe, and leu) confer a regulatory response primarily to a single cognate amino acid, whereas others
(thr, ilvGMEDA, and ilvBN) confer a multivalent response. In at
least one case (the ilvGMEDA operon in S. mal'cesens), a single
trp
Met Lys Ala Ile Phe Val Leu Lys Gly Trp Trp Arg Thr Ser OPA
AUG AAA GCA AUU UUC GUA CUG AAA GGU UGG UGG CGC ACU UCC UGA AACGGGCAGUGUAUUCACCAUGCGUAAAGC
---"----.-~..
~
+27
~'2
9
{p
his
Met Thr Arg Val Gin Phe Lys His His His His His His His Pro Asp AMB
AUG ACA CGC GUU CAA UUU AAA CAC CAC CAU CAU CAC CAU CAU CCU GAC UAG UCUUUCAGGCGAUGUGUGCUGGAAGACAUUCA
+32
----"---:;----1..
~
~'2'
{p
phe
Met Lys His Ile Pro Phe Phe Phe Ala Phe Phe Phe Thr Phe Pro OPA
AUG AAA CAC AUA CCG UUU UUC UUC GCA UUC UUU UUU ACC UUC CCC UGA AUGGGAGGCGUUUCGUCGUGUGAAACAGAAUGCGAAGACGAACAAU
..
~~.---~------~--~--~------
+22
1
pheST
Met Asn Ala Ala Ile Phe Arg Phe Phe Phe
2
TYr Phe Ser Thr OPA
AUG AAU GCU GCU AUU UUC CGC UUC UUU UUU UAC UUU AGC ACC UGA AUCCAGGAGGCUAGCGCGUGAGAAGAGAAACGGAAAACAGCGCCUG
+:;;
l' ..
,. ,
2
Met Lys Arg Ile Ser Thr Thr Ile Thr Thr Thr Ile Thr Ile Thr Thr Gly Asn Gly Ala Gly OPA
AUG AAA CGC AUU AGC ACC ACC AUU ACC ACC ACC AUC ACC AUU ACC ACA GGU AAC GGU GCG GGC UGA CGCGUACAGGA
"------,..---1..
~
+45
9,.
9M
2'
leu
Met Ser His Iie Val Arg Phe Thr Gly Leu Leu Leu Leu Asn Ala Phe Ile Val Arg Gly Arg Pro Val Gly Gly Ile Gin His OCH
AUG UCA CAU AUC GUU CGU UUC ACU GGG CUA CUA CUA CUC AAC GCA UUU AUU GUG CGC GGU AGA CCG
+27
0
ilvqMEDA
9
9
9
1
....
-iii
GU~GC
~o
0
2
AUU CAA CAU UAA
0
P
Met Thr Ala Leu Leu Arg Val Ile Ser Leu Val Val Ile Ser Val Val Val Ile Ile Ile Pro Pro Cys Gly Ala Ala Leu Gly Arg Gly Lys Ala ANB
AUG ACA GCC CUU CUA CGA GUG AUU AGC CUG GUC GUG AUU AGC GUG GUG GUG AUU AUU AUC CCA CCG UGC GGG GCU GCA CUU GGA CGA GGA AAG GCU UAG
..
,,.
9
'
{p
+33
ilvBN
2
Met Thr Thr Ser Met Leu Asn Ala Lys Leu Leu Pro Thr Ala Pro Ser Ala Ala Val Val Val Val Arg Val Val Val Val Val Gly Asn Ala Pro ANB
AUG ACU ACU UCC AUG CUC AAC GCA AAA CUA CUA CCA ACU GCG CCA UCC GCC GCA GUG GUC GUC GUG CGU GUG GUG GUG GUC GUC GGC AAU GCG CCG UAG
+32
..,..
2
tp.
FIGURE 6 Leader peptide coding regions and pause signals of eight operons regulated by a ribosome-stalling, alternative RNA structure mechanism of transcription attenuation. The RNA'sequences of the leader regions for E. coli tIp, S. typhil11uriul11 his, E. coliphe, E. coli pheST, E. coli tlu; S.
typhil11uriul11 leu, E. coli ilvGMEDA, and E. coli ilvBN were obtained from the GenBank DNA sequence
database. The locations of RNA secondary-structure elements (indicated by numbered horizontal
arrows) are shown as illustrated in reference 91. The locations of pause sites (P adjacent to vertical line
or arrow) are shown as reported for the tIP (171), his (25), thr (45), leu (14), ilvGMEDA (57), and
ilvBN (57) operons.
(
81
attenuator readthrough are observed when the cognate amino
acid is present or withdrawn from the medium. For instance,
such manipulation changes pheA operon expression 17-fold
(46).
Transcription-Translation Coupling. In principle, the coupling
of polymerase movement over the leader region to ribosome
positioning in the leader peptide coding region could be accomplished in two ways, stochastically or by pausing and release of
RNA polymerase. Synchronization could be achieved stochastically if the time between exposure of the leader peptide initiation
site and extrusion from RNA polymerase of the portion of RNA
segment 2 whose pairing with segment 1 or 3 dictates the attenuation decision were commensurate with the time required for
ribosome binding and initiation ofleader peptide synthesis. The
distance between the leader peptide start codon and this critical
portion of segment 2 varies from -50 to -75 nt (Fig. 6). At its
average rate over long transcriptional units (35 to 50 ntis) (21,
22, 167), RNA polymerase would require 1 to 2 s to complete
synthesis of this portion of leader RNA. This appears to be
sufficient time for stochastic synchronization with ribosome
movement (but see below). However, the available data favor the
pausing-release model for synchronization. Every leader region
examined to date contains a pause site at a location appropriate
to stop RNA polymerase before the critical portion of RNA
segment 2 is exposed (Fig. 6). Further, experiments performed
with a coupled in vitro transcription-translation system showed
that the paused complex decays much more slowly when leader
peptide synthesis is prevented by addition of inhibitors (88).
Nonetheless, direct demonstration of a requirement for pausing
to obtain synchronization has not yet been obtained because of
the complications that alternative RNA folding imposes on experimental design.
Basal-Level Expression. One interesting feature of attenuation
that has received some attention in recent years is the role of
basal-level readthrough of the attenuator even in the presence
of an adequate supply of cognate amino acid. The defining
observation of basal-level readthrough is the fivefold reduction
in tIP operon expression caused by the trpL29 mutation, which
changes the leader peptide initiation codon from AUG to AUA
(83, 187). In S. marcescens, deleting the tIP leader peptide start
codon causes a 10-fold reduction in operon expression (161).
Similar reductions in operon expression also have been observed
for leader peptide initiation codon mutations in the his (73) and
phe (46) operons. Although occasional slow movement of ribosomes through the leader peptide coding region even under
optimal conditions could explain this effect (91), at least for the
tIP operon the major cause of basal-level readthrough appears to
be the release of ribosomes at the leader peptide stop codon (Fig.
2) (138, 139). From the effects of several stop codon and ribo- .
some release factor mutants, Roesser and Yanofsky (139) suggest
that in tryptophan excess, 75% of the ribosomes remain attached
to the leader RNA at the stop codon while RNA polymerase
moves over the attenuator, thereby directing terminator formation. The remaining 25% of the ribosomes release, with the RNA
folding into terminator and antiterminator conformations with
equal probability. This produces 15% basal-level readthrough
when combined with the 3% readthrough inherent in the tIP
attenuator. This mechanism allows significant adjustment of
basal-level readthrough in different organisms by changes in the
TRANSCRIPTION ATTENUATION
1271
relative stabilities of the leader RNA termination and antitermination conformations (176).
Polymerase Recognition of Pause and Termination Signals. Until
recently, explanations for transcription pausing and Rho-independent termination have focused on the role of the nascent
transcript RNA hairpin. Findings obtained since the first edition
of this book was published suggest that these mechanisms, and
indeed the entire process of RNA chain elongation, may be
considerably more complex than had been appreciated (24,27,
31; chapter 55). Because the complexity of this subject is beyond
the scope of this review and because it is covered in another
chapter, we will offer only a summary of the most important
points regarding RNA chain elongation, pausing, and termination and then consider their implications for the mechanisms of
attenuation. The reader is referred to chapter 55 and the other
recent reviews cited above for more detail.
RNA chain elongation. Early. studies of in vitro transcription revealed that RNA chain elongation is discontinuous and
punctuated by long pauses at certain sites and rapid elongation
through other regions. It now appears that this variability in the
elongation reaction may reflect significant heterogeneity in the
structure of transcription complexes located at different template positions. Studies of isolated transcription complexes
halted at single template positions reveal significant differences
in the size of the DNA footprint on DNA (varying between -25
and 40 nt) (84, 90, 175), the position of the transcript relative to
the downstream edge of the complex (24, 90, 94,175), the size of
the single-stranded region of DNA within the complex (24, 94),
and the stability of individual complexes (85).
The pattern with which these properties change in two different promoter-proximal regions led Chamberlin (24) to propose
an "inchworm" model for discontinuous translocation of RNA
polymerase (see also references 19, 27, and 31). In this model,
RNA polymerase is envisioned to contain two separate binding
sites for DNA. These sites alternately lock and slide to allow
addition of up to 10 nt to the growing RNA chain while the
upstream contact moves forward and the downstream contact
remains fixe'd. When sufficient strain accumulates in the complex, the downstream contact unlocks and undergoes a discontinuous forward step of up to 10 bp. The RNA transcript also is
envisioned to be positioned on RNA polymerase in two distinct
sites. Approximately 8 nt at the 3' end of the RNA chain may be
paired to or positioned near the DNA template in a "loose"
RNA-binding site, such that the 3' end is near the leading edge of
the transcription bubble. The next 10 nt appear to remain tightly
bound to RNA polymerase, perhaps in a distinct transcript exit
channel. Filling of the loose site with RNA allows chain extension during the time the downstream contact is locked.
This model immediately raises several fundamental questions (27): (i) Is a constant inchworm cycle maintained as polymerase moves along the DNA? (ii) Are discontinuous changes
in RNA polymerase contacts to DNA or RNA somehow linked to
recognition of pause or termination signals? (iii) Do different
RNA polymerase molecules transcribe with precisely the same
discontinuous movements (i.e., remain in phase with each
other)? (iv) What is the effect of an upstream polymerase on
movement of a downstream polymerase? The answer to the first
question probably is no. Not only should this produce periodic
effects on transcript elongation patterns that have not been observed, but also it now appears that RNA polymerase behavior is
1272
LANDICK ET AL.
more monotonic over at least certain sequences located further
from the promoter (125). We address the second question below, but answers to the last two await further study (see also
reference 27).
Pause signals. Extensive studies of pausing in the his and trp
leader regions suggest that pause site recognition is complex and
relies on multiple interactions between RNA polymerase and
different segments of RNA or DNA. Rather than depending
simply on formation of the 1:2 (or A:B) secondary structure to
stop chain elongation, the his and trp pause sites are multipartite
and consist of at least four components (26,27, 95): (i) a 5- or
6-bp stem-loop RNA secondary structure (the pause hairpin) 10
or 11 nt upstream of the transcript 3' end, (ii) a 3'-proximal
region of transcript or DNA template, (iii) the apparently unfavorable geometry ofU or C at the 3' end of the transcript reacting
with an incoming GTP, and (iv) up to 14 bp of DNA downstream
of the pause site. Recognition of these pause sites appears to be
coupled to a discontinuous rearrangement of the transcription
complex at the pause site: the pause hairpin can be detected by
RNase VI digestion only at or 1 nt before RNA polymerase
reaches the pause site and occurs coincident with ;i 10-bp advance
in the downstream edge of the RNA polymerase-DNA contact.
However, the downstream edge of the complex remains fixed as
RNA polymerase approaches the pause site, apparently causing
the complex to compress as it passes over the 10 bp upstream
from the pause site (168). This compressed complex, which may
form either by movements of parts of the polymerase or by
looping of the RNA and DNA chains, isomerizes into a paused
configuration by a 10-bp jump in the downstream DNA contact
and simultaneous formation of the pause hairpin. The downstream sequence, and perhaps sequences just upstream from the
pause site, appear to set up isomerization into the paused configuration by controlling the inchworm movement of RNA polymerase. An altered downstream sequence causes more than
75% of the complexes to pass the site in vitro without entering
the paused state (168), probably because the requisite discontinuous movements in the complex have been compromised. The
pause hairpin, on the other hand, appears to stabilize the paused
configuration by an ionic interaction between the phosphate
backbone and a positively charged region on RNA polymerase
(26, 27). Whether this interaction slows elongation through an
allosteric effect on RNA polymerase or by altering the positioning
of the transcript in the active site remains to be determined.
Termination signals. Like pause sites, Rho-independent termination sites are multipartite (136). In addition to the terminator
RNA hairpin and following uridine-rich segment, the traditional
hallmarks of a Rho-independent terminator, both the DNA sequence downstream from the release site and the RNA or DNA
sequence upstream from the terminator hairpin also contribute to
the efficiency of termination for at least some Rho-independent
terminators with imperfect uridine-rich regions (e.g., UUUCUGCG [136,163]). These different interactions may explain the
terminator-specific effects of salt and nucleoside triphosphate
(NTP) concentration on termination efficiency (13 7). As we suggest
above for the pause site, precise and discontinuous movements in
the transcription complex also appear to contribute to the mechanism of termination (126, 168).
Outstanding Questions about Attenuation in Amino Acid Biosynthetic Operons. Although work on the mechanisms of attenuation in the amino acid biosynthetic operons of enteric
bacteria has slowed in recent years, it is not for lack of fundamentally important questions. Rather, these questions have become increasingly difficult to address experimentally. We outline here several among them that concern fundamental
macromolecular events that could fruitfully occupy talented
investigators in the years ahead.
Structures and dynamics of nascent transcript RNA folding.
First, what are the true stabilities in vivo of the alternative RNA
secondary structures that participate in attenuation control, and
even more important, how fast do they form once the relevant
segments of nascent transcript emerge from the transcription
complex? Do as yet unidentified proteins interact generally with
RNA and influence stability? Attenuation control relies on the
relative stability of RNA secondary structures, yet we know
surprisingly little about the precise conformations of these
structures, their true stabilities, their interactions, or the kinetics
of their formation. Although available enzymatic digestion data
support the presumed structures of the tIP leader transcript (Fig.
2; 86, 90, 128), many leader transcript structures have been
assigned simply on the basis of predictions of a computer algorithm. These algorithms do not accommodate the known complexity of RNA structures, which includes many different types
of non-Watson-Crick base pairs and tertiary interactions (173).
Thus, it is altogether possible that structures presumed for some
leader transcripts, particularly the more complicated ones like
those for ilvGMEDA (Fig. 5), may be missing important details.
Moreover, we are even less knowledgeable about the kinetics of
nascent transcript folding or of hairpin melting. For instance,
how fast after a ribosome releases from the leader peptide stop
codon does folding into the tip 1:2 or 2:3 structures occur? With
the increasing power and sophistication available to study structure with techniques like nuclear magnetic resonance spectroscopy, these questions should be approachable.
Role of transcription pausing: A second fundamental question concerns the role of transcription pausing in attenuation.
In the case of pyrimidine biosynthetic operons, the essential role
of transcription pausing to the mechanism appears indisputable
(see below). However, it is currently possible to argue that transcription and translation in the attenuator regions of amino acid
biosynthetic operons are synchronized either stochastically or
by the formation of a paused transcription complex and its
subsequent release by the ribosome translating the leader peptide coding region. Although several arguments favor the latter
possibility (see above), it has not been confirmed byexperimental observation. If pausing is required, elimination of the pause
signal or of the ability of RNA polymerase to recognize it should
prevent increased readthrough of the attenuator in response to
undercharged tRNAs or defective aminoacyl-tRNAs. This is not
simple to test experimentally, however, because most base
changes that would weaken the pause signal would also alter the
stability of the alternative RNA secondary structures, and because the pause signal is multipartite (see above). Elimination of
any single component of the pause signal has at most a five- to
sixfold effect on the pause half-life (26). Nonetheless, our greater
understanding of the pause signal now makes it possible to
design appropriate experiments. One could eliminate pausing
almost completely by combining selected base substitutions in
several components of the pause signal (particularly the DNA
sequence downstream from the pause site, which appears to be
critical for efficient recognition of the signal). These substitu-
81
[
f
l
lr
r
I
I':
I
i(
(
1
!
II
~,
I
I
I
tions could be inserted in attenuation control regions in otherwise isogenic strains with or without a mutation, such as miaA
or hisT, that causes elevated readthrough at theattenuator. If
transcription pausing is required to ensure ribosome participation within the critical window of time, theh elimination of
pausing should also diminate the increased readthrough caused
by l11iaA or hisT.
Relative timing of RNA polymerase movements. The essence
of attenuation control is the kinetic relationships among RNA
polymerase movement on DNA, ribosome movement on nascent
RNA, and RNA folding. It therefore follows that we will not understand the mechanism of attenuation completely until we understand the kinetics ofleader transcript formation and leader peptide
synthesis. Although the effects on attenuation of different ribosome
and RNA polymerase locations now are relatively clear (Fig. 3), the
actual timing of events during the movement of RNA polymerase
and the ribosome over the leader DNA and RNA are at present
simply best guesses. Assumptions about the time required for RNA
polymerase to reach the pause signal or for a ribosome to reach the
control codons, for instance, are based on average elongation rates
over long transcriptional or translational units (e.g., see kinetic
arguments in references 55 and 91). As the foregoing discussion of
RNA chain elongation, pausing, and termination should make
abundantly clear, the movements of RNA polymerase are complex
(see above). The average rate of transcript elongation, in fact, reflects
the rate-limiting influence of pause sites within a transcriptional unit
(3D, 79, 97). The rate of transcription between these pause sites is
uncertain but could be up to 10 times faster than the average. Hence
the apparent reasonableness of stochastic coupling between transcription and translation in a leader region (discussed above) or the
kinetic argument for the necessity of a double-ribosome model for
ilvGMEDA attenuation (discussed above) may be illusory. There is
no reason to assume that ribosome movements will be any less
complex.
A true understanding of attenuation will require that we explain
the behavior of each of these complex molecular machines in detaiL
Precisely how RNA polymerase moves through the leader region
matters a great deal to models of attenuation. One intriguing possibility, for instance, arises from the possible requirement for a particular coupling between discontinuous translocation by RNA
polymerase and recognition of the terminator. If RNA polymerase
is capable of transcribing the terminator in different phases of the
discontinuous cycle, it is conceivable that the transcription pause
also plays a key role in attenuation by establishing the configuration
of the transcribing complex so that it enters the attenuator region
properly phased for terminator recognition.
Evolution of attenuation in amino acid biosynthetic operons.
One intriguing question is why attenuation control in E. coli and
presumably S. typhil11uriu111 is used to regulate expression of
particular amino acid biosynthetic operons (tiP, his, phe, leu, tlll~
ilvGMEDA, and ilvEN) and an aminoacyl-tRNA synthetase operon (pheST) and not others that at least superficially appear
equally suitable (e.g., cysDNC, argCEH, or metELF). Interestingly, the attenuation-controlled, amino acid biosynthetic operons encode enzymes that catalyze assembly of related amino
acids (Trp, Phe, and His [aromatic] and Leu, Thr, He, and Val
[branched chain]). Perhaps the bacterium requires a particularly
rapid response to starvation for these particular amino acids
when its growth medium changes quicldyfrom one rich in these
particular amino acids (such as the human gut) to one in which
TRANSCRIPTION ATTENUATION
1273
they are absent. It is also possible that the energetic cost of synthesizing the aromatic and branched-chain amino acids makes it
desirable to maintain particularly efficient control over expression of their biosynthetic enzymes. Finally, one should not
disregard the simple possibility of historical accident leading to
retention or loss of attenuation control during divergent evolution of related operons.
Class II: Ribosome-RNA Polymerase Coupling
The first several examples of attenuation control discovered in E.
coli and S. typhil11uriul11 involved mechanisms that regulated the
expression of amino acid biosynthetic operons. Each example
employed ribosome stalling at control co dons as a regulatory
signal and an alternative transcript secondary structure as a
means of preventing terminator hairpin formation. These similarities raised the possibility that attenuation control was limited
to amino acid biosynthetic operons and to a single mechanism
for regulating transcription termination. The first clear indication to the contrary was the discovery of attenuation control of
pyrEl operon expression in E. coli (118, 142, 165). The pyrEl
operon encodes the pyrimidine biosynthetic enzyme aspartate
transcarbamylase, which is not involved directly in amino acid
metabolism. Elucidation of the pyrEl attenuation control mechanism revealed that transcription termination at the attenuator, a
Rho-independent transcription terminator that precedes the
pyrEl structural genes, was regulated in a way fundamentally
different from that described for the amino acid biosynthetic
operons (reference 89 and references therein); namely, transcription termination was controlled by the extent of coupling (i.e.,
movement and location) of a translating ribosome and a transcribing RNA polymerase within the pyrEl leader region. In this
case, the ribosome directly controls the formation of the terminator RNA hairpin by steric hindrance.
Shortly after the discovery of attenuation control of pyrEl expression, a similar mechanism was described for the pyrE gene of E.
coli (18, 130, 132), which also encodes a pyrimidine biosynthetic
enzyme. Attenuation control mechanisms equivalent to their E. coli
counterparts also appear to regulate pyrEl (114) and pyrE (120)
expression in S. typhil11uriul11. At present, the pyrEl operon and
pyrE gene provide the only examples of this second class of attenuation control, which we designate the ribosome-RNA polymerase
coupling mechanism; however, attenuation control of E. coli ampC
expression could be considered a special case (see below). In this
section, we will describe the general features of this class of attenuation control, provide a detailed description of the well-studied
mechanism of the pyrEl operon of E. coli, and comment on unique
features of pyrE regulation. The reader also is referred to an earlier
review (89) and to the chapter in this book on pyrimidine metabolism (chapter 35) for additional information.
Common Features and General Model. There are three regulatory elements required for the known examples of attenuation
control by ribosome-RNA polymerase coupling. These elements
are present in the leader region located between the promoter and
the first regulated structural gene (Fig. 7). These elements include
(i) a polypeptide-encoding open reading frame, which is referred to as the leader open reading frame; (ii) a Rho-independent
transcription tenhinator (attenuator) near the downstream end of
the leader openreading frame; and (iii) UTP-sensitive transcription
pause sites (runs of uridines in the leader transcript) that precede
the attenuator in the leader open reading frame. These elements
1274
LANDICKET AL.
Promoter-Leader Region
Promoter
Transcription
Pause Sites
/
R%8l
~
"
RS&1
Attenuator
Regulated Gene(s)
~
Leader Open Reading Frame
Readthrough
Transcription.
Low UTP - Strong Transcription Pausing
)
Leader Transcript
ppp----------------Transcription
Termination
~
,
Terminator
Hairpin
ppp---------FIGURE 7 General model for attenuation control by ribosome-RNA polymerase coupling (class II).
The diagram shows the relative positions of RNA polymerase and the translating ribosome (i.e.,
tightly coupled or uncoupled) when UTP levels are either low or high. Note that there is no requirement for ribosome binding or translation of the leader transcript at high UTP. (See text for additional
details.)
permit UTP-mediated regulation of gene expression according to
the following general model (Fig. 7). Transcription is initiated at a
promoter preceding the leader open reading frame and proceeds
into this region. When the intracellular level of UTP is low, RNA
polymerase stalls at UTP-sensitive pause sites, which provides time
for a ribosome to initiate translation of the leader transcript and
translate up to the stalled polymerase. When polymerase eventually
escapes the pause region and transcribes the attenuator, formation
of the terminator RNA hairpin is blocked by the presence of the
adjacent translating ribosome. In this case, polymerase continues
transcription into the downstream gene(s). In contrast, when the
level ofUTP is high, polymerase transcribes the leader region without stalling at UTP-sensitive pause sites. In this situation, there is
insufficient time for a ribosome to establish tight coupling with
polymerase before the formation of the terminator hairpin. The
result is transcription termination before the regulated structural
gene(s).
Attenuation Control of pyrBl Expression in E. coli. Most of what
we lmow about class II attenuation control comes from the study
of the pyrBl operon of E. coli. This operon encodes the catalytic
(pyrB) and regulatory (pyrl) subunits of the allosteric enzyme aspartate transcarbamylase, which catalyzes the first committed step
in the de novo synthesis of pyrimidine nucleotides. Expression of
the pyrBl operon is negatively regulated over an approximately
300-fold range by pyrimidine availability, specifically by the intracellular concentration of UTP (103, 104, 148, 164, 165). The first
indication that attenuation was involved in this regulation came
from the sequence determination of the pyrBl promoter-leader
region, which revealed a potential Rho-independent transcription
terminator (attenuator) located 23 bp before the pyrB structural gene
(118, 142, 165). Transcription from the pyrBl promoter (102)
was shown to be efficiently (98 to 99%) terminated at this attenuator
in vitro (165) and in vivo under conditions of pyrimidine excess
(96). The sequence of the 158-bp pyrBIleader region indicated that
the leader transcript could not adopt alternative stem-loop structures to regulate terminator hairpin formation, which implied that
attenuation control of pyrBl expression must be mechanistically
different from that described for the amino acid biosynthetic operons (165).
Elucidation of the pyrBl attenuation control mechanism relied
on the discovery of two additional regulatory elements. The first
81
TRANSCRIPTION ATTENUATION
that removes the run of eight T·A base pairs at the end of the pyrBl
attenuator plus one additional base pair to maintain the reading
frame of the leader polypeptide (104). All Rho-independent transcription termination is abolished at this mutant attenuator.
When the mutant strain was grown under conditions of pyrimidine excess, pyrBl expression was approximately 50-fold higher
than that in an isogenic pyrBr strain. When growth of the mutant
was limited for pyrimidines, operon expression increased an
additional sevenfold. Growth of the pyrBI+ strain under the same
pyrimidine-limiting conditions resulted in a 300- to 350-fold
increase in operon expression (103,104). These results indicate
that attenuation control is responsible for most (i.e., 50-fold), but
not all, of the pyrimidine-mediated regulation. Recent studies
indicate that the residual sevenfold pyrimidine-mediated regulation in the mutant strain is due to an additional pyrBl control
mechanism. In this mechanism, high UTP levels induce reiterative transcription within a run of three T·A base pairs in the
initially transcribed region, which inhibits promoter clearance
(103).
Translation of the pyrBl leader transcript. In the model for
pylEl attenuation control, translation of the 44-codon open reading
frame of the leader transcript plays the critical role of disrupting the
formation of the terminator hairpin. It was necessary, therefore, to
demonstrate that such translation occurs physiologically. This task
was accomplished by fusing the pyrBl promoter region and leader
open reading ii'ame to the lacZ gene and detecting the predicted
was a translatable 44-codon open reading frame that extends
through the leader region and ends 6 nt before the pyrB gene (Fig. 8)
(141). The second was strong UTP-sensitive transcription pause sites
in the pyrBIleader region upstream of the attenuator (36, 165) (Fig.
8). These features suggested a regulatpry model (as drawn in Fig. 7)
in which low UTP levels cause RNA polymerase to transcribe slowly
through the first half of the leader region, which results in tight
coupling between polymerase and a ribosome that is translating the
44-codon open reading frame. When polymerase eventually transcribes the attenuator,' the terminator hairpin is precluded from
forming or is disrupted by the adjacent translating ribosome. The
result 'is transcription into the pyrBTstructural genes and synthesis of
aspartate transcarbamylase when it is needed by the cell.
To describe the regulatory elements more clearly and to illustrate the interactions required for class II attenuation control, we
will discuss in detail the key features of the E. coli pyrBl control
mechanism.
Attenuation versus attenuation-independent control of
pyrBl expression. An early question about the pytEl attenuation control mechanism was whether it could account for all
pyrimidine (i.e., UTP)-mediated regulation of operon expression. This regulation occurs over a much wider range than the
typical 10- to 40-fold range for attenuation control of amino acid
biosynthetic operon expression in E. coli and S. typhimurium
(91). To answer this question,pyrBl expression was measured in
a mutantE. coli strain which carries a 9-bp chromosomal deletion
Pause Hairpin
ACAAUUU
G
Lys
A
60A AA
10 Cc
G
A Lys
Leu U=A
CsG
U=A Asp
Arg GsC
CsG A~
Pro GSC 7&,y
U
U Met Val Gin Cys Val Arg His Phe Val Leu CsG Leu Pro Phe Phe Phe
GUAUGGUUCAGUGUGUUCGACAUUUUGUCUUACs
GCCUGCCGUUUUUCUUC,
,
G
G
G
A
G
X
I
,
,
20
30
50
40
80
~,
Terminator Hairpin
Asn A U
A
C120
CsG Arg
Leu U=A
C=G
-
CsG Gly
Pro CsG
C
Gln 110 =G Ala
Pro Leu lie Thr His Ser G=CPhe Phe Cys Pro Gly Val Arg Arg Stop
Met
CCGUUGAUCACCCAUUCCCA = UUUUUUUUGCCCAGGCGUCAGGAGAUAAAAG AUG
90
100
1275
130
140
150
pyrB
FIGURE 8 Nucleotide sequence and secondary structures of the pyrBl leader transcript. Transcription initiation occurs at the first two A residues in vitro and almost exclusively at the second A in vivo.
Nucleotides 21 through 152 encode the 44-amino-acid leader polypeptide, and the sequence ends
with the AUG initiation codon of the pyrB cistron. The Shine-Dalgarno sequences for the leader
polypeptide and pyrB are underlined. Two RNA secondary structures 'are shown; the first (pause
hairpin) is flanked by uridine-rich sequences, within which strong UTP-sensitive transcription pausing occurs, and the second (terminator hairpin) is specified by the pyrBl attenuator. The sites of
transcription termination at the pyrBl attenuator correspond to positions 133 to 135.
1276
LANDICK ET AL.
leader polypeptide-~-galactosidase fusion protein in cells (141).
However, it is not the 44-amino-acid leader polypeptide that is
important for regulation but the act of translation of the leader
transcript. Ihis fact was demonstrated by showing that near-normal
attenuation control was observed in a mutant bearing a +1 frameshift at codon 6 of the 44-codon open reading frame, which still
allows translation of the entire leader region (28). This situation is
comparable to that described above for the leader peptides of amino
acid biosynthetic operons.
To show that regulation requires translation of the 44-codon
leader open reading frame, the effects of mutations that either
strongly inhibit the initiation of translation of the leader transcript or introduce stop co dons early in the open reading frame,
well before the attenuator, were measured (28, 140, 141). Each
mutation reduced operon expression to ~6% of the wild-type
level under conditions of pyrimidine limitation and to ~30% of
the wild-type level under conditions of pyrimidine excess; the
latter effect presumably reflects a low level of tight coupling of
transcription and translation within the wild-type leader region
even in cells grown with an ample pyrimidine source. The net
effect of the mutations was to significantly reduce the range of
pyrimidine-mediated regulation. Further support (or the regulatory role of translation of the leader transcript comes from a
study of pylEl (and pyrE) expression in cells in which ribosomes
translate at either one-third or two-thirds of their normal rate
because of a mutation in the IpsL gene encoding the ribosomal
protein S12 (69). When mutant cells were grown with excess
pyrimidines, they exhibited significantly reduced pyrBl expression compared to the wild-type level. Apparently, the slower rate
of translation reduced coupling of transcription and translation
in the leader region, resulting in increased transcription termination at the attenuator. In contrast, the slow ribosomes did not
significantly affect pyrBl expression under conditions of mild or
severe pyrimidine limitation, indicating that in these situations,
transcription pausing within the leader region is sufficient to
permit extensive coupling of transcription and translation even
with slow ribosomes.
The proposed attenuation control model employs a translating ribosome to physically block the formation of the terminator RNA hairpin under conditions of pyrimidine limitation. To
test this aspect of the model, the distance that a ribosome must
translate within the leader region to suppress transcription termination at the attenuator was determined by introducing stop
co dons at various sites in the leader open reading frame from
co dons 6 to 33 (140). On the basis of the estimated size of the
ribosome-binding site (77, 156), translation would have to proceed to within approximately 15 nt of the terminator hairpin to
permit the ribosome to interact directly with this sequence. The
results show that translation termination at or before codon 24,
which is 16 nt upstream of the terminator hairpin (Fig. 8), reduces
operon expression to approximately 5% of the wild-type level under pyrimidine-limiting conditions. In contrast, when translation
termination occurs at codon 25, which should be the first stop
codon at which ribosome-binding overlaps the sequence of the
terminator hairpin, expression is 64% of the wild-type level. In
general, the level of operon expression increases as the stop codon is
moved further downstream of codon 25, with the highest level of
expression (i.e., 91 % of the wild-type level) occurring with translation termination at codon 33, which is within the loop of the
terminator hairpin. In S. typhimuriul11, the leader open reading
frame normally stops at codon 34 as a result of a sequence
difference between the two bacteria (114). These results provide
strong support for the proposed regulatory role of the ribosome.
According to the model for regulation, a single round of
translation of the leader transcript is sufficient to elicit readthrough transcription. Consistent with this limited requirement
for translation, the ribosome-binding site preceding the leader
open reading frame is relatively weak, only 7% as efficient as the
pyrB ribosome-binding site (K. 1. Roland, C. Liu, and C. L.
. Turnbough, Jr., unpublished data). Interestingly, mutations in
the leader ribosome binding site that increase leader translation
by as much as lO-fold cause less than a 2-fold increase in operon
expression under conditions of pyrimidine excess, and they have
no significant effect on operon expression under conditions of
pyrimidine limitation (c. Liu and C. 1. Turnbough, Jr., unpublished data). Thus, the pylEl attenuation control mechanism
appears fine-tuned to produce strong regulation without unnecessary leader translation.
Additional control of translation in the leader region is suggested by the presence of sequences between nt 75 and 143 of the
leader transcript (Fig. 8) that are complementary to the leader
ribosome-binding site (1l8, 141). Formation of a secondary
structure by these sequences could block multiple rounds of
translation of readthrough transcripts and perhaps all translation of attenuated transcripts. It is also possible that this secondary structure plays a more active role in attenuation control.
For example, in cells grown under conditions of pyrimidine
excess, rapid transcription up to the attenuator may be quicldy
followed by the formation of a secondary structure which precludes ribosome binding to' the leader transcript and thereby
ensures transcription termination at the attenuator. Such a role
remains to be established experimentally.
VTP-sensitive transcription pausing in the pyrBl leader
region. The discovery of VTP-sensitive transcription pausing
in the pyrBl leader region was a key finding in the development
of the attenuation control model. This pausing provided the
regulatory sensor, equivalent to the control codons in the case of
the amino acid biosynthetic operons, that could respond to different levels of VTP in a way that controlled transcription termination at the attenuator. DIP-sensitive pause sites are defined as
positions in RNA (or DNA) atwhich RNA polymerase stalls while
awaiting the addition of a uridine residue to the 3' end of a
growing RNA chain. Generally, these pause sites are detectable
only at low VTP concentrations (e.g., below 200 11M). In E. coli
and S. typhim urium, the VTP concentration varies from approximately 50 11M in cells grown under conditions of pyrimidine
limitation to 1 mM or slightly above in cells grown under conditions of pyrimidine excess (6, 119, 164).
The first in vitro experiments to detect VIP-sensitive transcription pausing in the pyrBIleader region revealed only a small
cluster of pause sites that correspond to a uridinecrich region
located 20 nt before the terminator hairpin in the leader transcript (Fig. 8) (165). Subsequent in vitro studies employing
more accurate and sensitive procedures have provided a significantly different view of pausing in the leader region preceding
the attenuator (36). Instead of one cluster of pause sites, there
are a large number of sites throughout the leader transcript at
which RNA polymerase pauses when the VTP concentration is
low (i.e., 20 11M with other NTPs at 400 11M). Nearly all of these
sites correspond to positions where uridines are added to the
r
81
leader transcript. Pausing at these sites decreases with increasing
UTP concentrations al}d is no longer detectable at a concentration of 400 f.tM. Although some degree of pausing apparently
can occur before the addition of every uridine residue in the
leader transcript at 20 f.tM UTP, the str~ngth of individual pause
sites is variable. This variability presumably reflects the effects of
DNA sequence and RNA secondary structure (25, 95). In this
regard, strong pausing occurs before the addition of uridines at
leader transcript positions 81 to 85, which are located 7 nt
downstream of an RNA hairpin (designated the pause hairpin in
Fig. 8). These sites correspond to the originally identified cluster
of UTP-sensitive transcription pause sites. Recent studies of
hairpin-induced transcription pausing suggest that the pyrEl
pause hairpin would affect specifically the rate of transcript
elongation through positions 81 to 85 in the leader region (26,
27). A mutation that destabilizes the pause hairpin has in fact
been shown to reduce pausing in this region (K. Mixter-Mayne
and C. 1. Turnbough, Jr., unpublished data).
Although some pause sites within the leader region may be
stronger than others, the large number of these sites indicates
that it is the cumulative effect of pausing at multiple positions
that is the key factor in controlling coupling between RNA polymerase and the ribosome translating the pyrEl leader transcript. Consistent with this view, replacing all seven uridines
with adenines between positions 81 and 88 in the leader transcript (Fig. 8) causes only a twofold reduction in tIte range of
pyrimidine-mediated regulation of pyrEl expres~ion (MixterMayne and Turnbough, unpublished data). Int~restingly, this
large substitution mutation causes an approximately lO-fold
increase in the level of readthrough transcription at the pyrEl
attenuator in vitro (from 2 to 20%). This result may indicate
that strong UTP-sensitive pausing at positions 81 to 88 in the
wild-type leader transcript is used to establish a transcription
complex configuration that is optimally phased for subsequent
attenuator recognition as discussed above.
Unlike transcription at low UTP concentrations, appreciable
transcription pausing in the pyrEl leader region is not detected
at 20 f.tM ATP or CTP and is detected at only one site at 20 f.tM
GTP (36). Strong pausing occurs before the addition of a guanine residue at position 55 in the leader transcript (Fig. 8). This
pausing is substantially reduced, although not completely eliminated, at 400 f.tM GTP. The reason for pausing at this site and
not before the addition of other guanine residues in the leader
transcript remains to be established, but presumably it is associated with the function of a secondary structure in the transcript.
Although nucleotide G-55 is included in the pause hairpin of the
leader transcript (Fig. 8), its position in the 5' segment of the
stem indicates that the observed pausing is unrelated to the
formation of this hairpin. The physiological consequences of
pausing at this unique GTP-sensitive site are not known. However, pyrEl expression is increased fourfold in cells defective in
guanine nucleotide synthesis (68). Possibly, this effect is du~ to
GTP-sensitive transcription pausing that permits coupling of
transcription and translation in the pyrEl leader region 'and
readthrough transcription past the pyrEl attenuator. '
The more pronounced pausing at 20 f.tM UTP than at 20 f.tM
ATP, CTP, or GTP appears to be due, at least in part, to a
difference in the apparent KI1l values for these nucleotides during
transcript elongation. The apparent KI1l for UTP during elongation appears to be significantly higher than the apparent Kill
TRANSCRIPTION ATTENUATION
1277
values for the other NTPs (70, 82). A high~r apparent KI1I for
UTP and the fact that UTP (unlike CTP) pools fall to low levels
in cells limited for pyrimidines (131) make UTP an ideal regulatory effector for attenuation control based on ribosome-RNA
polymerase coupling.
Additional data supporting the proposed role of UTP-sensitive transcription pausing in attenuation control come from
studies of a strain of S. typhimurium carrying an altered RNA
polymerase that exhibits an approximatety sixfold-higher apparent Kill for the binding of UTP (6 mM) and ATP (4 mM) during
transcript elongation. This mutant displays constitutive expression of the pyrEl operon and the pyrE gene at high intracellular levels of UTP (70), indicati~g that transcription
pausing during the addition of uridine (or other) residues to
the pyrEl leader transcript, and not the UTP level per se, is
the key determinant in regulation. Furthermore, the transcript elongation factor NusA enhances UTP-sensitive pausing within the pyrEl and pyrE leader regions in vitro (6, 36) and
appears to be important in determining the level of expression
of these genes in vivo (6). Presumably, NusA plays a key role in
establishing a rate of transcript elongation that permits tight
coupling of transqiptiOJ:i and translation in cells limited for
pyrimidines. It has been shown that pyrE expression is hyperrepressed by the addition of uracil to a culture of E. coli containing a defective NusA (6). Apparently, transcript elongation is too
fast in the mutant strain to permit coupling of transcription and
translation in the pyrE attenuator region, particularly in cells
containing high levels of UTP. These results indicate that the
activity of NusA or of any factor that influences the rate of
transcript elongation can affect the expression of genes regulated
by class II attenuation control.
Unique Features of pyrE Attenuation Control. The pyrE gene
of E. coli encodes the pyrimidine biosynthetic enzyme orotate
phosphoribosyltransferase. Expression of this gene is regulated over a 30-fold range almost entirely by an attenuation
control mechanism which is analogous to that described for
the pyrEl operon (18, 69, 130, 131). A striking difference,
however, is that the pyrE "leader open reading frame" is 238
co dons long and encodes a functional protein. This open
reading frame is actually the rph gene, which encodes the
tRNA processing exoribonuclease RNase PH (127). Apparently, the pyrE gene is the second gene of a bicistronic operon,
and the cell uses transcription and translation of the first
cistron for the purpose of attenuation control of pyrE expression.
UTP-sensitive as well as GTP-sensitive transcription pausing has
been demonstrated in the Iph transcript (6). Also noteworthy is
that the Iph region of the transcript ends eight bases before the
terminator RNA hairpin specified by the pyrE attenuator. However, on the basis of the size of a typical ribosome-binding site,
translation to the en~ of the Iph cistron would result in efficient
disruption of th~ terminator hairpin, thereby allowing readthrough transcription.
Outstanding Questions. Perhaps the most intriguing question
at present is whether this general class of attenuation control,
which was the first to "broaden our attenuation horizons," is
actually very lirpited itself. For e4a~ple, is it limited to pyrimidine genes because only changes in UTP pools can affect transcription pausing appropriately? Alternatively, this class of attenuation control may be more prevalent and based, in some
1278
LANDICK ET AL.
cases, on changes in the activity of other factors that control
coupling of transcription and translation.
~II
Class III: Regulatory Factor Dependent
READTHROUGH
•
In the two classes of attenuation control described so far, regulation of transcription attenuation is achieved by coupling transcription and translation within the regulatory leader region. In
the third class, called regulatory factor-dependent attenuation,
the regulatory role of translation is replaced by a trans-acting
RNA-binding factor. This factor can be a protein or an antisense
RNA that binds a specific sequence in the leader (or intercistronic) region of the regulated transcript. Factor binding can
either directly block the formation of an attenuator-specified
terminator hairpin or indirectly control its formation by favoring
a particular RNA secondary structure.
bglOperon. In E. coli, the best studied example of regulatory
factor-dependent attenuation is regulation of bgl operon expression. In this case, the regulatory factor is a specialized RNA-binding protein. The bgl operon contains three genes, bglG, bglF, and
bglB, which enable cells to use certain aromatic ~-glucosides such
as salicin and arbutin as carbon sources (133, 147). The bglG gene
encodes a positive regulatory protein, BglG, which is required for
operon expression. The bglF gene encodes the ~-glucoside-spe­
cific enzyme n of the phosphoenolpyruvate-dependent phosphotransferase system (designated enzyme nBg1 or BglF), which
is present in the cytoplasmic membrane and transports ~-glu­
cosidic sugars into the cell with concomitant phosphorylation of
the sugar. The bglB gene encodes phosphb-~-glucosidase B,
which hydrolyzes phosphorylated ~-glucosides to yield glucose6-phosphate. The bgl operon is cryptic in wild-type cells, but a
variety of spontaneous mutations activate the operon by enhancement of transcription initiation from ~ preexisting but
silent promoter (105). Once the operon is activated, full expression requires a ~-glucoside inducer and also cycli~ AMP (cAMP)
and the cAMP receptor protein (CRP) (135).
~-Glucoside-mediated regulation of bgl operon expression occurs through an attenuation control mechanisj11 (Fig. 9). In the
absence of a ~-glucoside inducer, most transcripts initiated at the
activated bgl promoter are terminated at a Rho-independent attenuator located just upstrea~ of bglG, the first gene in the operon
(109). A second Rho-independent attenuator is located between
bglG and the adjacent bglF gene (147). The positive regulator BglG
is required to prevent transcription termination at both attenuators
(109, 145). Low levels of BglG and BglF are synthesized in the
absence of ~-glucosides, but under these conditions, BglF inactivates BglG by phosphorylation (2, 3,146). Phosphorylation ofBglG
prevents the formation of dimers, which are required for RNA
binding and antitermination activity (4). In the presence of ~-glu­
cosides, BglF dephosphorylates BglG, allowing it to dimerize and
function as an antitermination factor (2,3, 146). Nonphosphorylated dimers of BglG prevent transcription termination within the
operon by binding to a sequence in the bgl mRNA that precedes and
partially overlaps the terminator hairpin encoded by each attenuator. This binding blocks the formation of the terminator hairpins,
allowing expression of the operon. Apparently, the BglG-binding
site forms an alternative secondmy structure that is recognized by
BglG dimers (5, 60).
At present, it is not known if S. typhimuriul11 carries· a bgl
operon. Attempts to clone parts of the operon by PCR have been
unsuccessful. S. tJphimuriul11 does not grow on aromatic ~-glu-
bg/
Allenualor
Promoler
1
Attenuatar
2
bglG
~
... ~
~Active
Phospho-
bglF
TERMINATION
~
...
8+8lnaclive
p
p
~-glucoside
Membrane
~-glucoside
~-glucoside present,
8glG dephophorylated
by 8g1F.
No ~-glucoside,
8glG phosphorylated
by 8g1F.
FIGURE 9 Model for attenuation control of bgl operon expression
showing the effects on transcription of BgIF-catalyzed phosphorylation or dephosphorylation of BgIG in the absence or presence,
respectively, of ~-glucoside inducer. (See text for additional details.)
cosides, indicating that if the genome includes a bgl operon, it is
silent (A. Wright, personal communication).
Antisense RNA-Mediated Control of Plasmid Replication. At present, there are no clear cases of antisense RNA-dependent attenuation in E. coli, but this type of regulation can be illustrated by
examining the mechanism that controls replication of the staphylococcal plasmid pTl81 (124). An analogous mechanism controls
replication of the streptococcal plasmid pIP50 1 (20). Replication
of plasmid pTl81 is controlled by the level of the initiator protein
RepC. Constitutive transcription of plasmid pTl81 produces an
antisense RNA capable of base pairing with a target sequence near
the 5' end of the repC transcript. When this pairing occurs early
during repC transcription, it promotes the formation of an RNA
hairpin 5' to the repC start codon. This hairpin causes premature
Rho-independent transcription termination, which precludes
RepC synthesis. In the absence of the antisense RNA (i.e., when
plasmid copy number is low), an upstream sequence in the repC
leader transcript pairs with the 5' segment of the terminator
hairpin, thereby blocking the signal for transcription termination
and allowing synthesis of the full-length repC transcript. In this
case, RepC is synthesized and can direct another round of plasmid replication.
Class IV: Rho Factor Dependent
p. coli and S. typhimuriul11, and perhaps most bacteria, have two
distinct classes of transcription termination sites. One class, discussed previously, is responsible for termination in many operons regulated by transcription attenuation. Sites of this class
appear to be recognized by RNA polymerase acting alone; they
are referred to as Rho-independent or intrinsic termination sites.
Sites of the second class require the action of the protein factor
Rho. Rho interacts with both transcript and RNA polymerase and
directs polymerase to terminate transcription (chapter 55). The
first example of the use of transcription termination and its relief
in gene reguliltion ipvolved Rho factor and its role in regulating
early gene transcription in bacteriophage lambda. The phagespecified N protein was shown to be responsible for antitermination at Rho-dependent termination sites (53).
Sl
Much is known about Rho's structure and,mechanism of
action (23, 47, 53; chapter 55). The characteristics of many
Rho-binding sites and Rho termination sites have been deter. mined. Rho has been shown to be an ATP-dependent RNADNA helicase (23); helicase activity is required for termination.
Rho causes terminatiop. by contacting a paused RNA polymerase molecule and altering its behavior. Rho action requires
accessory factors, such as NusG (chapter 55). Models that explain Rho's binding to RNA, its relative movement on RNA, and
the mechanism by which it promotes termination have been
proposed (23,47). The end of an operon is often defined by a
Rho-dependent termination site. Occasionally Rho termination.
sites are located preceding or within a structural gene, where
they allow Rho to participate in regulatory events that control
gene expression. Rho is involved in transcription attenuation
regulation in the tna (15S), livL (170), and rRNA (155) operons,
and it also modulates its own synthesis (113).
Rho-Dependent Transcription Attenuation in the tna Operon. The
tna operons of E. coli and Proteus mirabilis are regulated by
tryptophan-induced transcription antitermination (52, 75,
157-159). These operons contain two major structural genes:
tnaA, encoding the hydrolytic enzyme tryptophanase, and tnaB,
specifying a tryptophan permease (34, 75). The presence of these
two proteins permits these organisms to use tryptophan as a
carbon or nitrogen source. In both organisms, tnaA is preceded
by a leader regulatory region containing a short coding region,
tnaG, encoding 24- and 36-residue leader peptides in E. coli and
p. 111irabilis, respectively (Fig. 10) (75, ISS). tnaG and tnaA are
separated by a ca. 100- to 200-bp region that contains regulated
sites of Rho-dependent transcription termination (75, ISS). A
third tna operon, that of Enterobacter aerogenes SM-lS, is organized similarly (SO). S. typhi111uriu111 does not have a tna operon.
Transcription initiation in the tna operons of E. coli and P.
111irabilis, as in many degradative operons, is subject to catabolite
repression. A cAMP-CRP-binding site is located just upstream
of the -35 site in each tna promoter. Activated CRP presumably
binds at these sites and promotes initiation. Transcription initiation is not subject to tryptophan control. Rather, the presence of
exogenous tryptophan induces an antitermination mechanism.
that prevents Rho-dependent termination in the leader region
of the operon (157, ISS). Mutational studies have shown that
Rho alterations that reduce its termination proficiency increase
basal tna operon expression (157, ISS). Similarly, deletions that
remove a presumed "rut" (Rho utilization) site located immediately following tnaG, or that remove segments of the spacer
segment between tnaG and tnaA, also increase basal operon
expression (52; A. Kamath and C. Yanofsky, unpublished data).
Studies on the role of tnaG in tna operon induction in E. coli
and Proteus vulgaris have shown that translation of tnaG is 'essential for tryptophan-induced antitermination. Thus, mutations that alter the tnaG start codon eliminate tryptophan
induction (52, 159). tnaG of these two organisms contains
single Trp codon, which, when replaced by other co dons, prevents ~nduction by tryptophan or by the amino acid encoded by
the substituted codon (52). Replacing the Trp codon by a stop
codon also prevents induction. However, introducing an appropriate tRNATrp nonsense suppressor gene, but not other tRNA
suppressor genes, restores induction (52). Frameshift mutations
that allow translation of tnaG to proceed beyond its stop codon
result in elevated, constitutive expression of the operon (52,
a
TRANSCRIPTION ATTENUATION
tnaC
.
~I
(I
co~
...;~
internal stop
~UGG-UGA """""'"
Trp codon
rut site
1279
tnaA
..........
Rho
termination
sites
ill!
basal expression; no extracellular Trp
W.C
reading
frame 2
p ......-.
U'tJ
~UGA
ribosome binds and
translates in rf2
and dissociates at
rf2 UGA
J.
tnaA
l,.
,
"Rho binds
and signals RNP
to terminate
transcription
Trp-induced expression
~j.
tnaA
I)
I
FIGURE 10 Proposed mechanism of regulation of the tna operon.
(Top) Organization of the tna leader region, including tnaC and the
following spacer region that contains sites of Rho-dependent transcription termination. Features of the transcript that are essential
for regulation are the in-frame UGG (Trp) codon and out-of-frame
UGA (stop) codon, in tnac. Also shown is the approximate location
of an RNA segment, rut, presumed to be required for Rho binding.
(Middle) Molecular events believed to be resp~nsible for the low
basal-level expression of the operon in cells growing without extracellular tryptophan. (Bottom) Induction by tryptophan and the
proposed role for the TnaC leader peptide in preventing Rho binding to the tna transcript. Translation of the single Trp codon in tnaC
by tRNATrp is essential for induction.
ISS). In addition, when the tna leader transcript is highly expressed from a multicopy plasmid, induction of the resident tna
operon is reduced appreciably (50), suggesting that some cell
component becomes limiting for antitermination.
Recent mutational studies have provided evidence suggesting
that the TnaC leader peptide itself plays a role in tryptophan-induced antitermination (K. Gish and C. Yanofsky, unpublished
data). Nonconservative amino acid changes at six positions in
TnaC prevent induction, whereas conservative changes at these
same positions, or silent changes in co dons specifying the residues at these positions, have no effect on induction (Gish and
Yanofsky, unpublished data). However, with the exception of six
consecutive amino acid residues of TnaC that include the single
Trp residue, the TrpCs from the three bacterial species have very
1280
LANDICKET AL.
different amino acid sequences. This observation suggests that
some or all of these six residues may be crucial in promoting
antitermination.
Among the mutational changes in tnaC ~f E. coli that have
been examined, one important class results in high-level constitutive expression of the operon. Mutations of this class convert
an out-of-frame stop codon near the middle of tnaC (Fig. 10) to
anyone of several sense codons. In these mutants, tna operon
expression is five times higher than the tryptophan-induced
level observed with the wild type (Gish and Yanofsky, unpublished data). An out-of-frame stop codon is located at a similar
position in tnaC of the tna operons of the other two bacterial
species.
On the basis of studies with the tna operons of E. coli and P.
vulgaris (Gish and Yanofsky, unpublished data; Kamath and
Yanofsky, unpublished data), a tentative model that explains
many features of this example of transcription attenuation can
be proposed (Fig. 10). According to this model, segments of the
tna leader region have two crucial functions. One is to allow Rho
to bind to the transcript and cause RNA polymerase to terminate transcription. This is the principal event that must occur in
cells grown in the absence of tryptophan. This function is believed to be mediated by the out-of-frame stop codon within
tnac. Presumably, ribosome stoppage or dissociation at this stop
codon allows Rho to recognize and bind to the following untranslated segment of mRNA. The second function of the leader
region is to permit an unidentified tryptophan induction event
to provide an activated leader peptide (i.e., TnaC) that prevents
Rho molecules from attaching to the leader transcript (perhaps
by inhibiting ribosome release), hence allowing expression.
A boxA-like sequence has been identified at the end of tnaC
in the E. coli and P. vulgaris operons (158; Kamath and Yanofsky,
unpublished data), raising the possibility that some RNA-binding accessory factor binds here and influences Rho's action. The
precise binding site for Rho in the tna leader transcript is not
known, nor is it known how tryptophan promotes antitermination. This example, like others in which Rho is a participant, is
complex.
rRNA Operons. A special case of Rho-dependent attenuation
occurs in the seven homologous rRNA operons of E. coli and S.
typhi111uriu111, which encode a 16S-23S-5S rRNA precursor from
which the mature rRNAs are excised nucleolytically (see chapter
90). The elongating rRNA transcript, unlike other nascent RNAs,
is not translated and thus is a potential target for Rho protein
throughout its synthesis. To prevent Rho from stopping rRNA
synthesis, the cell has evolved a special mechanism, termed rRNA
antitermination. The degree to which this process is regulated is
not known. We will describe rRNA antitermination here because,
although it is related to N- and Q-dependent antitermination
during bacteriophage lambda growth (see chapter 55), it is the
only clear-cut example of a cellular regulatory mechanism that
alters the capacity of RNA polymerase to elongate an RNA chain
through an entire transcriptional unit.
Transcription initiation in rRNA (1'/'11) operons occurs at two
promoters, PI and P2, that couple the rate of initiation to
growth rate, stringent response, and translational capacity of the
cell (see chapter 90). Between the start site and the first nucleotide of the mature 16S rRNA, the rRNA transcripts contain two
sequences, boxA and boxB, that are important for antitermination and that also are present in the nut regions of phage
lambda, where they participate in N-dependent antitermination
(chapter 55). boxB encodes an RNA stem-loop structure. boxA
encodes the consensus sequence 5'-UGCUCUUUAACA, which
appears to be a target for binding of a heterodimer of NusB and
ribosomal protein SID (NusE) (Ill, 122). Interestingly, the order of these sequences in the 1'/'11 leaders is boxB-boxA, opposite
that found in the lambda nut sites. Mutations in boxA and NusB
abrogate RNA antitermination (100, ISO, 155). Although deletion of boxB does not prevent antitermination, some boxB muta. tions do, perhaps because boxB can bind both an inhibitor and
an activator of antitermination (155). The boxB-boxA motif is
highly conserved among both eubacteria and archaebacteria
(15), suggesting that rRNA antitermination may be a universal
mechanism except in eukaryotes, in which a special RNA polymerase has evolved for rRNA synthesis.
rRNA antitermination has been recapitulated in vitro. In
addition to RNA polymerase, an 1'/'11 template, NusB, NusA,
NusE (SlO), and NusG, rRNA antitermination in vitro requires
an unidentified cellular protein that may be the 1'/'11 homolog of
the phage lambda N protein (155).
Several other features of the rRNA antitermination mechanism raise interesting questions that are worthy of further study.
First, rRNA antitermination blocks only Rho-dependent, not
Rho-independent, termination (1). In fact, tandem Rho-independent terminators halt transcription at the end of the I'm
operons. How this is accomplished and why it differs from the
anti termination mechanisms involved in lambda gene expresssion are not known.
Second, the involvement of a ribosomal protein, SlO (NusE),
in rRNA antitermination raises the as yet unexamined possibility of a feedback control circuit in which excess 16S rRNA inhibits antitermination by sequestering SID.
Third, by analogy to lambda N-dependent antitermination,
the boxB-boxA region of the 1'/'11 nascent RNA is thought to be
anchored to antitermination-modified RNA polymerase as it
transcribes the I'm genes (112, 121). This finding raises the possibility, suggested first by Morgan (115), that the 5' stem of the
"processing stalk," which is required for excision of 16S rRNA, is
delivered to the 3' stem by the transcribing polymerase. Such a
mechanism also could account for the existence of a second
boxA downstream from the 16S gene, where it could facilitate a
second antitermination modification of RNA polymerase, after
cotranscriptional excision of 16S rRNA, and possibly set up 23S
processing by a similar delivery mechanism.
Finally, the finding that fivefold-faster transcription of I'm
gen~s by T7 RNA polymerase produces inactive ribosomes (98)
raises the possibility that a precise elongation rate is necessary
for proper folding of rRNA (155). Thus, by determining the
time available for 5'-proximal segments of the rRNA t9 fold
. before potentially competing 3' -proximal segments are synthesized, r RNA antitermination, by itself doubling the normal elongation rate, may control the sequence of events by whi<;;h rRNA
reaches its proper three dimensional conformation.
Other Interesting Examples
Ribosomal Protein L4 Mediates Transcription Attenuation in the
Leader Region of the SlO Operon of E. coli. The SID operon of E.
coli contains the structural genes for 11 ribosomal proteins. Four
of these proteins are constituents of the 30S ribosomal subunit,
while the remaining seven are constituents of the 50S ribosomal
)
I
81
subunit (71; chapter 90). The SID operon, like other operons
encoding ribosomal proteins, is autogenously regulated by one
of its specified proteins. This regulatory protein, L4, is encoded
by the third gene of the operon (71; chapter 90). L4 is one of
several primary rRNA-binding proteins that recognize'specific
segments of 23S rRNA during the initial stages of assembly of the
50S ribosomal subunit (152).
The L4 protein is believed to regulate SID operon expression
by two distinct mechanisms, translational feedback 'inhibition
and transcription attenuation (for a recent review, see reference
185). Feedback inhibition occurs whenever synthesis of L4 provides a molal' excess of this protein over 23S rRNA. The free L4
molecules are believed to bind to the SID transcript and inhibit
translation of the SID coding region (101, 180). Inhibition of
translation of the SID coding region also reduces translation of
the downstream coding regions in the SID transcript. Thus, SlO
synthesis and L4 synthesis are translationally coupled (70, 101).
Translational feedback inhibition is a common regulatory feature of ribosomal protein operons of E. coli, i.e., ribosomal
proteins specified by genes of other ribosomal protein operons
also autogenously regulate their own synthesis. These proteins
presumably also act by binding to their homologous transcripts
(70). Binding ofL4 to the SID transcript during the initial stages
of the transcript's synthesis also appears to reduce transcription
of the structural genes of the operon, via transcription attenuation (42, 181). It has been shown that some mutations in the
SID leader region influence translational control, others alter
transcriptional control, while some mutations affect both processes (42). These findings suggest that the regulatory sites essential for the two processes have both unique and common
features.
The L4 protein regulates SID operon transcription by promoting transcription termination (attenuation) in the SID
leader region (42, 181). The extent of attenuation control in vivo
by L4 is about fourfold. Here, as in translational control, when
L4 is present in excess over 23S rRNA, it functions as a regulator.
The binding site for the L4 protein in 23S rRNA has been localized to a lID-base segment designated domain I (184). The
presumed L4-binding site(s) in the leader segment of the SlO
transcript that is responsible for attenuation control has not
been determined (149). Transcript sequences and structures that
are somewhat similar to sequences and structures in 23S rRNA
have been identified in the vicinity of the ribosome-binding site
used for SID synthesis (180). The L4-S10 example is currently
the only one in which transcription attenuation regulates expression of a ribosomal protein operon. As mentioned earlier,
rRNA synthesis in E. coli also is subject to transcription attenuation (155).
L4-mediated transcription termination has been studied in
vivo and in vitro. Under both conditions, L4 promotes transcription termination at a typical Rho-independent termination
site located 140 bp from the transcription start site of the SID
operon (182). Translation of leader RNA is not involved in
operon regulation. Rather, it appears that L4 recognizes some
feature of the transcript 01' transcription complex during transcription of the leader region of the operon (183). The NusA
protein is required for transcription termination at the SID attenuator in vitro (183). NusA is believed to act by increasing the
stability of a paused transcription complex that forms at the SID
leader termination site. The NusA-facilitated paused complex is
TRANSCRIPTION ATTENUATION
1281
thought to be stabilized further by association with the L4 protein (149, 183); this complex then presumably promotes transcription termination at the attenuator. Although attempts to
demonstrate direct binding of L4 to the SID leader transqipt
have been unsuccessful (149,184), mutational studies have identified two segments of the leader transcript that form hairpin
structures as being essential for L4 action (149, 183). The L4
protein promotes transcription termination in vitro when added
to a NusA-RNA polymerase complex that has reached a pause
site (183). This observation suggests that L4 recognizes the
NusA-mediated paused complex. However, although ability to
form the attenuator hairpin is sufficient for NusA action, an
RNA segment upstream of this hairpin is necessary for L4 stabilization of the paused complex (183). This finding suggests that
additional sites or factors are necessary for L4 action. When
these sites and factors are discovered, and their mechanisms of
action are elucidated, it may be possible to describe the precise
events involved in L4-mediated transcription attenuation.
Transcription Attenuation in the ampC Operon of E. coli. The
gene ampC of E. coli encodes a ~-lactamase that is produced in
small amounts and is secreted into the periplasm, where it can
hydrolyze penicillin and related ~-lactam antibiotics (123). Expression of ampC is subject to an interesting form of growth rate
control (67). As the growth rate increases, the rate of production
of the ampC-encoded ~-lactamase also increases (54). Transcription of the ampC operon is initiated 41 bp preceding al11pC, in
the frd gene, which resides in a separate operon (16). The leader
segment of the al11pC transcript contains a typical Rho-independent transcription terminator (66). This transcript segment
also has a ribosome-binding site just upstream of the terminator
hairpin. The AUG start codon of this ribosome-binding site is
immediately followed by an ochre stop codon (66). A ribosome
bound at this ribosome-binding site would be expected to disrupt
the terminator, thereby allowing transcription to proceed into the
ampC coding region (66). On the basis of what is known about
ampC operon regulation, the following model has been proposed
to explain growth rate control. As the number of ribosomes per
cell increases or decreases, coincident with changes in growth
rate, it becomes more or less lilzely that a ribosome will bind at
the leader ribosome-binding site and disrupt the terminator.
Upon disruption of the terminator, synthesis of the ampC transcript and its specified periplasmic ~-lactamase would be increased. Thus, the ribosome content per cell would determine the
rate of transcription of ampC. Much evidence that is consistent
with this model has been gathered. Transcription studies performed in vitro have demonstrated formation of the expected
terminated transcript. Mutational studies have shown that ribosome binding and formation of the terminator hairpin are both
essenti~l for growth rate regulation (54, 66).
TRANSCRIPTION ATTENUATION MECHANISMS
IN OTHER ORGANISMS
Although this review deals with attenuation mechanisms in
E. coli and S. typhimuriul11, we think it essential that the reader
be aware of several recent discoveries of novel attenuation
mechanisms in other organisms, including both gram-negative
(11, 37, 40) and gmm-positive (13, 29, 33,49) bacteria. Similar
mechanisms could well be found in future studies of gene regulation in E. coli and S. typhimurium. Interestingly, these examples
are principally class III mechanisms (i.e., regulatory factor-de-
1282
LANDICKET AL.
pendent attenuation), and they involve some operons whose
homologs in E. coli and S. typhimurium are regulated by class I
and class II mechanisms.
B. subtilis trp Operon
Attenuation control of the trp operon in Bacillus subtilis, as in E.
coli, involves formation in the leader transcript of alternative
secondary structures that either include or exclude a terminator
hairpin (87, 151). However, in B. subtilis, formation of the transcript secondary structures is controlled not by the position of a
translating ribosome but by the binding of a regulatory protein,
TRAP (tIP RNA-binding attenuation protein), which is encoded
by the mtl'B gene (10, 51). Under conditions of abundant tryptophan, TRAP binds to a segment of the leader transcript upstream of the terminator hairpin. This binding blocks formation
of an antiterminator secondary structure equivalent to the E. coli
2:3 hairpin, thereby promoting formation of the terminator
hairpin and ~ausing transcription termination. Recent studies
indicate that TRAP consists of 11 identical8-kDa subunits (7, 8)
and that each tryptophan-activated subunit binds one closely
spaced G/UAG repeat in the leader transcript (10).
B. subtilis pyl' Operon
A similar mechanism also appears to regulate the pyl' operon in
B. subtilis. This operon encodes the six pyrimidine nucleotide
biosynthetic enzymes and includes two additional,S' -proximal
genes: pyl'R, which encodes a 20-kDa regulatory RNA-binding
protein that also can catalyze the conversion of uracil and phosphoribosylpyrophosphate to UMP, and pyrP, which encodes an
integral membrane uracil permease (166). Distinct Rho-independent terminators (attenuators) occur in the leader region, the
pyrR -PYI'P intercistronic region, and the pyrP-pyrB intercistronic
region. Recent studies suggest that when pyrimidines are abundant, the PyrR protein binds to target sequences in the nascent
transcript and disrupts formation of antiterminator secondary
structures that, when the pyrimidine pool is depeleted, prevent
formation of the terminator hairpins (106). An intriguing feature
of this mechanism is the use of an enzyme involved in pyrimidine
metabolism as the RNA-binding protein, perhaps allowing the
binding site for UMP to be used both in the enzymatic reaction
and as an allosteric site to regulate the RNA-binding activity of
PyrR (R. Switzer, personal communication).
tRNA-Directed Transcription Antitermination
An exciting recent discovery is the finding that transcription
attenuation can be directly controlled by binding of a cognate
uncharged tRNA to the nascent leader transcripts of at least 12
aminoacyl-tRNA synthetase and 6 amino acid biosynthetic genes
and operons in a variety of gram-positive bacteria (reference 59
and references therein). These leader RNAs each contain three
conserved stem-loop structures (I, II, and III), followed by a
highly conserved 14-base element (T box) and then the attenuator-specified terminator hairpin. An unpaired triplet sequence
(called the sEecifier sequence), which corresponds to a codon for
the cognate amino acid, occurs in a bulge region of each stem I.
For all genes, an antiterminator hairpin can be formed by base
pairing of a segment of the T box with a conserved sequence in
the upstream portion of the terminator hairpin. In the antiterminator hairpin, the central seven bases of the T box form a
bulge. When uncharged cognate tRNA is abundant, it presum-
ably binds to the specifier sequence in stem I through codon-anticodon interactions and, near its 3' end, to the T-box bulge in the
antiterminator hairpin. This stabilizes the antiterminator hairpin
and permits readthrough transcription into the downstream
gene(s). When the cognate tRNA is charged, it either does not
bind stably or is unavailable for interaction because it is complexed with aminoacyl-tRNA synthetase or elongation factors.
This allows the terminator hairpin to form and cause transcription termination. Thus, limitation of a particular amino acid or
. aminoacyl-tRNA synthetase increases the level of uncharged cognate tRNA(s), which in turn increases transcription of the genes
encoding the enzymes that synthesize this amino acid or that
charge it to tRNA.
Possibility of Transcription Attenuation in Eukaryotes
Although our review focuses on bacterial attenuation mechanisms, it is worth mentioning the possibility that transcription
attenuation occurs in eukaryotes. Although similarities of features in eukaryotic transcriptional units to those found for bacterial attenuatots have led to several suggestions of transcription
attenuation in eukaryotes (38, 58, 1l0), to date there is no documented example of a eukaryotic gene whose expression is regulated at a discrete transcription termination site. However, in at
least some cases (the human and murine c-mycproto-oncogenes,
for instance), the susceptibility of RNA polymerase II to multiple,
weak termination sites within or upstream from a gene appears
to be controlled by events at or near the promoter (recently
reviewed in references 76 and 172). These mechanisms are most
formally analogous to bacterial antitermination mechanisms,
such as those found in rRNA operons (see above and chapter 90)
or during growth of phage lambda (chapter 55). For example, the
lambda Nand Q proteins play roles somewhat similar to that of
human immunodeficiency virus type 1 Tat protein, whose binding to a promoter-proximal RNA structure (the transactivation
response region) is required for efficient synthesis of full-length
mRNA (recently reviewed in reference 74).
Although other types of attenuation mechanisms have not
been found in eukaryotes, we caution against concluding they
do not exist. An important lesson from studies of bacterial attenuation is. that regulatory mechanisms evolve special features
that uniquely couple transcription of a particular gene to the
metabolic need for its gene product. Control of RNA chain
elongation in eukaryotes is poorly understood, and these linkages may still be unrecognized. Both class III mechanisms and,
as we have noted elsewhere (89), coupling of transcription by
RNA polymerase II to assembly of the mRNA splicing machinery (in analogy to coupling to ribosome movement in bacteria)
could plausibly occur in eukaryotes.
EVOLUTIONARY ASPECTS, CONCLUSIONS,
AND EXPECTATIONS
We have described a variety of transcription attenuation mechanisms that are used by E. coli and/or S. typhimurium in regulating
expression of specific operons. The diversity of these mechanisms
and the variety of molecules and events involved lead us to
suspect that there are many as yet undiscovered mechanisms of
. transcription attenuation used by bacteria. Potential targets for
regulatory events that could modulate transcription termination
are RNA polymerase, polymerase accessory proteins, RNA-binding proteins, ribosomal components, polypeptide and ribosome
81
release factors, termination factors, and the transcripts themselves. Thus, as mentioned in the introduction, the many pote~r
tial targets for regulation by transcription attenuation probably
offer cells options that are not available for regulation of transcription initiation. A second consideration is the impact of
evolution on adoption of successful regulatory strategies. lfRNA
did serve as the sole genetic material during some early period,
as some suggest, and if this RNA was translated by a primitive
translational process, then some of the transcription attenuation
mechanisms in use today could have evolved from regulatory
mechanisms that were operating prior to the appearance of DNA.
It seems likely that successful strategies would have been retained.
As we have mentioned, there are only a few well-documented
examples of transcription attenuation in eukaryotes. The patterns of gene expression in higher organisms suggest that a
common regulatory objective is to provide each genetic unit
with a variety of cis regulatory sites or elements that allow an
appropriate response to numerous regulatory proteins that are
produced or· modified during different stages of gro~th and
development. Give)) the need for regulatory diversity, it seems
likely that some forms of transcription attenuation will be found
to be common in eukaryotes.
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