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Transcription and RNA processing Lecture 7 Biology 3310/4310 Virology Spring 2017 It is possible that Nature invented DNA for the purpose of achieving regulation at the transcriptional rather than at the translational level —ALAN CAMPBELL Parvovirus Hepatitis B virus VII Retrovirus Adenovirus Herpes simplex virus Polyoma- and Papillomaviruses Transcription Reovirus Rotavirus Poliovirus Influenza virus Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Eukaryotic DNA-dependent RNA polymerases Enzyme Cellular RNA Viral RNA RNA pol I Pre-rRNA None known RNA pol II Pre-mRNA Pri-miRNA SnRNA Pre-mRNA Pri-miRNA HDV genome RNA and mRNA RNA pol III Pre-tRNAs 5S rRNA U6 snRNA Ad-2 VA RNAs EBV EBER RNAs MHV68 pri-miRNA Only DNA viruses that replicate in cytoplasm (poxvirus, giant viruses) encode an RNA pol Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University Transcription is the first biosynthetic reaction to occur in cells infected with dsDNA viruses Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Parvovirus Hepatitis B virus VII Retrovirus Adenovirus Herpes simplex virus Polyoma- and Papillomaviruses Transcription Reovirus Rotavirus Poliovirus Influenza virus Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Conversion of viral genomes to templates for transcription Which viral genomes do not need conversion? Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Transcription is regulated Binds TFIID Specifies accurate starts (Position and orientation independent) Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University (Specific DNA sequences that bind proteins) ©Principles of Virology, ASM Press Regulatory sequences in transcriptional control regions Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press up to 10,000 bp away! Enhances initiation! Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Proteins that regulate transcription • • Host and/or virus sequence-specific DNA binding proteins • Many co-activators modulate structure of nucleosomal templates Viral co-activating molecules (do not bind DNA but can modulate transcription) also required Crosslink proteins to DNA Isolate chromatin Anti-H Input Immunoprecipitate ©Principles of Virology, ASM Press Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University Purify DNA Bead Amplify herpesviral DNA sequence Sequence-specific transcriptional activators Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Stimulation of transcription by Ad E1A proteins Histone deacetylases remove acetyl groups from histones, wrap DNA more tightly, reducing transcription Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Go to: b.socrative.com/login/student room number: virus What is the first biosynthetic event that occurs in cells infected with dsDNA viruses? A. B. C. D. E. Membrane fusion Transcription DNA replication Protein synthesis All of the above 1 Strategies of transcription of viral DNA Origin of transcriptional components Virus Host only Simple retroviruses Host plus one viral protein that regulates transcription Complex retroviruses, parvoviruses, papillomaviruses, polyomaviruses Host plus >1 viral protein that stimulate transcription Adenoviruses, herpesviruses Viral Poxviruses Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University Regulation of transcription by viral proteins Positive autoregulatory loop (may also be negative) Cascade regulation Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Viral transcriptional programs: SV40 Early Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University Late ©Principles of Virology, ASM Press Regulation of SV40 late promoter by cellular repressors ibp = initiator binding protein Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Adenovirus transcriptional regulation • Three viral proteins and DNA synthesis govern phase transitions • E1A necessary for transcription of all E transcription units (frees E2f) • E2 required for DNA synthesis and entry into L phase, increases initiation from major late promoter • IVa2 enhances L gene transcription Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Adenovirus transcription units Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Herpesvirus transcriptional programs • Initiated by VP16, a virion associated protein (differs from Py, Ad) • Activates IE transcription • IE proteins control transcription from all virus genes • Expression of E genes and DNA synthesis • Expression of L genes, DNA dependency • Ensures coordinated production of DNA genomes and structural proteins Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Go to: b.socrative.com/login/student room number: virus Adenovirus E1A protein stimulating the expression of adenovirus E2 protein which then stimulates the expression of adenovirus IVa2 & L4 protein is an example of: A. B. C. D. A negative autoregulatory loop Repression of gene expression Cascade regulation Dimerization 2 Modification of mRNA: 5’-cap structure Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press as reoviral and arenaviral mRNAs, may survive by virtue Figure 10.3 Model of cotranscriptional capping of RNA polymerase II transcripts. The elongating RNA polymerase II pauses after 20 to 30 nucleotides have been incorporated into a nascent transcript. The C-terminal domain (CTD) of the largest subunit is then phosphorylated (P) at specific serine residues. Such modification is the signal for binding of the capping enzyme to the CTD and capping (blue) if the nascent RNA. Co-transcriptional capping Initiation Incorporation of 20–30 nucleotides RNA polymerase II CTD phosphorylation Capping enzyme P P Synthesis of 5' cap 5' c Capping enzyme P P Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University RNA polymerase II are invariab lular enzymes (Table 10.2). Tran lular gene by RNA polymerase I at which poly(A) will be added. determined by endonucleolytic c a specific position. Such cleavage nation of transcription. Poly(A) terminus, while the RNA down is degraded (Fig. 10.5). Cleavage are identified by specific sequen simian virus 40 and adenovirus mature mRNA is formed 10 to 3 of a highly conserved and esse nal, the sequence 5′AAUAAA3′. not sufficient to specify poly(A is found within mRNAs at intern used as polyadenylation sites. S the cleavage site, notably a U- or 5 to 20 nucleotides downstream, mRNAs (particularly viral mRN 5′ to the cleavage site are also im The first reaction in polyadeny 5′AAUAAA3′ sequence by the p age and polyadenylation specifi This interaction is stabilized by Poly(A) polymerase is then recru to cleavage of the pre-mRNA b polymerase then synthesizes a p 200 to 250 nucleotides in a two©Principles of Virology, ASM Press Modification of mRNA: Cleavage and polyadenylation ~200A Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Addition of poly(A) to viral mRNAs Mechanism Enzyme Viruses Cellular Adenovirus, HBV, HDV, herpesviruses, polyomavirus, retrovirus Reiterative copying at stretches of U in template RNA Viral Influenza virus, VSV Copying of long U stretch in template RNA Viral Poliovirus, alphavirus Post-transcriptional Cleavage of pre-mRNA followed by polyadenylation During mRNA synthesis Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University Discovery of mRNA splicing in adenovirus infected cells Ad DNA (ss) Ad mRNA Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Splicing of pre-mRNA Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University spliceosome = ribozyme ©Principles of Virology, ASM Press Constitutive and alternative splicing Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University Viral proteins can regulate alternative splicing ©Principles of Virology, ASM Press Go to: b.socrative.com/login/student room number: virus Which statement about polyadenylation of DNA virus mRNAs is correct? A. B. C. D. It always occurs in the cytoplasm It occurs after cleavage of pre-mRNA Poly(A) is added at the 5’-end of pre-mRNA Is specified by a stretch of U residues in the template 3 Splicing marks mRNAs for nuclear export components of nuclear export pathway Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Export of unspoiled retroviral mRNA CTE = Constitutive transport element Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Rev protein regulates export of HIV mRNA Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Splicing = Value added • • • Alternative splicing creates different mRNAs, proteins Coding information of a small DNA genome is expanded Regulation of gene expression Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University DNA genomes Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press Why does transcription occur first for DNA viruses? Viral DNA replication always requires synthesis of at least one viral protein, sometimes many - hence it is always delayed after infection Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
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