Download 99 Bernard Lec 1

Bio99A: Molecular Biology, Spring 2010
Part 2: Gene expression / transcription
Hans-Ulrich Bernard (Uli Bernard)
Administrative issues:
Instructor: Dr. Uli Bernard,
Dept. Molecular Biology and Biochemistry + Progr. Publ. Health
Office: 114 Sprague Hall
Preferred Contact: discussion after lecture
Other possibilities: visit my office Friday 11-12 a.m.
(1/2 mile SW from here)
Email: hbernard@uci.edu
(but I cannot answer 400 emails per week)
I studied in
Göttingen, Germany,
apologies for my
accent !
I worked for 15 years
at the National
University of Singapore,
where I gave similar
lectures as this one,
but this is my first
Bio99 lecture at UCI.
This means: I know the science,
but I am still learning about
the student population.
Education systems vary:
I passed elementary and high school, college and graduate school
without taking a single multiple choice test.
As a father of high school kids, I hate tests and I hate cramming.
However, if you are in this room, you are here to become a biologist,
and you really have to know this stuff.
I will use about 80% material from “Tropp”, but part of the content
is from elsewhere or by my own design.
The test will be based on my slides, but read “Tropp” if you want to
know more.
Overall outline of Bio99:
Section 1: DNA and RNA, structure and enzymology
Section 2: Organization of genes and genomes
Expression of genes (transcription)
our topic
Section 3: Expression of genes (translation)
Individual topics:
Lecture 1: Genes, genomes, gene expression. What is transcription initiation?
Lecture 2: Promoters and RNA polymerases in prokaryotes
Lecture 3: Lac operon, negative regulation
Lecture 4: Lac operon, positive regulation, trp operon
Lecture 5: Promoters and RNA polymerases in eukaryotes
Lecture 6: Eukaryotic transcription factors and their binding sites
Lecture 7: Regulated factors, response elements
Lecture 8: Transcription in specific organs, differentiation, cancer
Lecture 9: Histones and chromatin, epigenetic regulation of transcription
Lecture 1: Genes, genomes, gene expression etc.
Lecture 1:
Genes, genomes, gene expression,
What is transcription initiation?
Lecture 1: Genes, genomes, gene expression etc.
What is “gene expression”?
Central concept “dogma” of molecular biology:
Transcription + translation leads from storage form
of genetic information (DNA) to functional proteins.
Lecture 1: Genes, genomes, gene expression etc.
What is “gene expression”?
Transcription + translation leads from storage form
of genetic information (DNA) to functional proteins.
This lecture series is about transcription.
Lecture 1: Genes, genomes, gene expression etc.
Terminology: What is a gene?
(simple model, appropriate for prokaryotes)
Coding sequences plus flanking elements involved in expression.
Lecture 1: Genes, genomes, gene expression etc.
Terminology: What is a gene ?
(complex model more appropriate for eukaryotes)
mRNA
Exons (coding sequences)
Introns (intervening sequences)
Transcription
+ splicing
5’ flanking region
= 5’ non-coding region
= upstream region
contains regulatory
elements
Promoter region
3’ flanking region
= 3’ non-coding region
= downstream region
determines transcription termination
can contain regulatory elements
These segments have to
be identified by sequence analyses
and in functional studies,
to detect meaning in a seemingly
featureless DNA double helix.
Lecture 1: Genes, genomes, gene expression etc.
What is an open reading frame (ORF) ?
A segment of DNA that can encode a polypeptide sequence, i.e.
is not interrupted by a termination codon. The term is used in
pro- and eukaryotes and does not require the presence of an ATG.
What is a cistron?
An ORF in prokaryotes which DOES contain an ATG. An mRNA can
have one cistron (monocistronic) or several (polycistronic).
What is a gene ?
Broader meaning than ORF and cistron. Definition can include
flanking regulatory sequences. Also applies to non-coding sequences
like rRNA and tRNA
What is an operon?
A set of genes, 2 or more, that are regulated and expressed together
on a single mRNA (polycistronic mRNA). The genes in an operon
have related functions.
Lecture 1: Genes, genomes, gene expression etc.
Some number games:
Size of proteins: Average 300 amino acid residues
(but wide range of less than 100 to more than 1000)
Average size of genes: 3 x 300 or 900 bp.
Bacterium like E. coli has about 4300 genes
= 4 million bp = actual genome size.
All DNA is needed for coding of proteins.
Humans have about 30,000 genes = 30 million bp,
but the haploid human genome is 3 billion bp.
Lots of junk in the human genome
(remember, “junk” is not “garbage”).
Lecture 1: Genes, genomes, gene expression etc.
What is a genome ?
Sum of all genes plus non-coding sequences !
Genome size in nucleotide pairs for various organisms:
about 4000 genes
about 30,000 genes
Lecture 1: Genes, genomes, gene expression etc.
Gene + Genome properties of pro- vs. eukaryotes
Prokaryotes: - most genes are contiguous, no introns
- many transcription units have multiple genes
(= cistrons organized in operons)
- short non-coding regions
- loose association of DNA and proteins
- genome located in cytoplasm, forms nucleoid
Eukaryotes: - many genes are not contiguous, have multiple exons
- most transcription units have single gene
- most DNA is non-coding
- tight and complex association of DNA and proteins
(= histones, form chromatin)
- genome located in nucleus, transcription occurs
in nucleus, translation in cytoplasm
Lecture 1: Genes, genomes, gene expression etc.
Gene + Genome properties of pro- vs. eukaryotes
Prokaryotes: - most genes are contiguous, no introns
- many transcription units have multiple genes
(= cistrons organized in operons)
- short non-coding regions
- loose association of DNA and proteins
- genome located in cytoplasm, forms nucleoid
Eukaryotes: - many genes not contiguous, have multiple exons
- most transcription units have single gene
- most DNA is non-coding
- tight and complex association of DNA and proteins
(= histones, form chromatin)
- genome located in nucleus, transcription occurs
in nucleus, translation in cytoplasm
Lecture 1: Genes, genomes, gene expression etc.
Irrespective of pro- or eukaryote, genes can be on either strand
of a double-stranded DNA
Transcript map of protein coding transcripts in Adenovirus
Lecture 1: Genes, genomes, gene expression etc.
Gene + Genome properties of pro- vs. eukaryotes
Prokaryotes: - most genes are contiguous, no introns
- many transcription units have multiple genes
(= cistrons organized in operons)
- short non-coding regions
- loose association of DNA and proteins
- genome located in cytoplasm, forms nucleoid
Eukaryotes: - many genes not contiguous, have multiple exons
- most transcription units have single gene
- most DNA is non-coding
- tight and complex association of DNA and proteins
(= histones, form chromatin)
- genome located in nucleus, transcription occurs
in nucleus, translation in cytoplasm
Lecture 1: Genes, genomes, gene expression etc.
Prokaryotic genomes are very compact:
- very little space between genes
- very little unfunctional (“junk”) DNA
- existence of “operons”
Lecture 1: Genes, genomes, gene expression etc.
Eukaryotic genomes are not compact
Lecture 1: Genes, genomes, gene expression etc.
Gene + Genome properties of pro- vs. eukaryotes
Prokaryotes: - most genes are contiguous, no introns
- many transcription units have multiple genes
(= cistrons organized in operons)
- short non-coding regions
- loose association of DNA and proteins
- genome located in cytoplasm, forms nucleoid
Eukaryotes: - many genes not contiguous, have multiple exons
- most transcription units have single gene
- most DNA is non-coding
- tight and complex association of DNA and proteins
(= histones, chromatin)
- genome located in nucleus, transcription occurs
in nucleus, translation in cytoplasm
Lecture 1: Genes, genomes, gene expression etc.
Gene + Genome properties of pro- vs. eukaryotes
Prokaryotes: - most genes are contiguous, no introns
- many transcription units have multiple genes
(= cistrons organized in operons)
- short non-coding regions
- loose association of DNA and proteins
- genome located in cytoplasm, forms nucleoid
Eukaryotes: - many genes not contiguous, have multiple exons
- most transcription units have single gene
- most DNA is non-coding
- tight and complex association of DNA and proteins
(= histones, form chromatin)
- genome located in nucleus, transcription occurs
in nucleus, translation in cytoplasm
Lecture 1: Genes, genomes, gene expression etc.
Localization of genome and gene expression
in prokaryotes versus eukaryotes
Lecture 1: Genes, genomes, gene expression etc.
Why study regulation of gene expression?
Bacterium: 4000 + genes
Higher organism: 30,000 genes
At any given time only a subset of these genes is expressed.
And this subset determines:
physiological properties
morphological properties
differentiation,
health and disease,
etc. etc.
Lecture 1: Genes, genomes, gene expression etc.
Gene expression:
Be aware of the dimensions of the undertaking:
1014 cells, each
with the same
30.000 genes
regulated
gene expression
Perfect human being
Lecture 1: Genes, genomes, gene expression etc.
Imagine the challenge of gene expression:
Even a small bacterial genome has thousands of genes,
and is not just a circle but a really long string.
The length of the DNA in each human cell is about 2 meters,
100,000 times the diameter of the cell where it resides.
Lecture 1: Genes, genomes, gene expression etc.
Why study regulation of transcription ?
Gene expression = Transcription + translation
But it is probably fair to say that the lion share of
regulation of gene expression occurs on the level of transcription.
Lecture 1: Genes, genomes, gene expression etc.
Why study regulation of initiation of
transcription?
The term “transcription” includes
- initiation
- elongation
- termination
and is linked to splicing, transcript stability etc.,
But, again, the lion share of regulation of transcription
occurs on the level of
regulation of transcription initiation.
Therefore, most of my lecture series is about regulation
of transcription initiation.
Lecture 1: Genes, genomes, gene expression etc.
Mechanistic concept of transcription:
Coding strand of DNA: the strand that corresponds
to the RNA used to translate the protein, running 5’ to 3’.
The template strand of the DNA
is transcribed to become the mRNA.
coding strand
5’
3’
non-coding strand
3’
5’
DNA
transcription
5’
coding strand
3’
RNA
Lecture 1: Genes, genomes, gene expression etc.
What are transcription and transcription initiation?
Lecture 1: Genes, genomes, gene expression etc.
Transcription starts with 5’ end of RNA
What does 5’ end of RNA mean?
Lecture 1: Genes, genomes, gene expression etc.
Transcription starts with 5’ end of RNA
What does 5’ end of RNA mean?
3’C of terminal nucleotide
(and all subsequent nucleotides)
becomes target of polymerization.
5’C of terminal nucleotide
(with attached triphosphate)
forms end of mRNA
Mechanism of polymerization:
Nucleophilic attack of 3’ OH
on alpha-phosphorylgroup of
Incoming NTP.
Lecture 1: Genes, genomes, gene expression etc.
Mechanism of transcription:
Nucleophilic attack of 3’ OH on alphaphosphorylgroup of incoming NTP.
5’
3’
5’
3’
Lecture 1: Genes, genomes, gene expression etc.
Site of transcription initiation is called a “promoter”
Promoter:
Region of DNA required for transcription initiation.
Lecture 1: Genes, genomes, gene expression etc.
Function of promoter:
Nucleotide sequences recognized by RNA polymerase to bind DNA
and initiate transcription.
RNA polymerase is the enzyme that transcribes a gene into a mRNA.
RNA polymerase
Lecture 1: Genes, genomes, gene expression etc.
A promoter is the site where
RNA polymerase binds DNA
and starts to transcribe a gene.
Different promoters of
bacteria
or of humans
have very different properties.
And this is what the next lectures
will be about !
Lecture 1: Genes, genomes, gene expression etc.
Summary
- Genes are nucleotide sequences that most often encode proteins
plus the flanking regulatory regions
- Transcription is the process to turn a DNA sequence into an RNA
- The enzyme transcribing genes is called RNA polymerase
- A promoter is the nucleotide sequence at the 5’ end of a gene required to
initiate transcription
- The DNA is read from the 3’ to the 5’ direction, the mRNA grows 5’ to 3’