Chapter 7: Synthesis of RNA from DNA Templates

Synthesis of RNA from DNA Templates Introduction During the infectious cycles of viruses with DNA genomes- viral messenger RNA (mRNA) synthesis must precede production of proteins. In most cases, this step is accomplished by the host cell enzyme that produces cellular mRNA, RNA polymerase II In contrast, viral RNA polymerases transcribe the large DNA genomes of viruses that replicate in the cytoplasm, such as poxviruses, but these enzymes resemble their host cell counterparts in several respects In most cases, the genes of viral DNA templates are expressed in a defined reproducible sequence: enzymes and regulatory proteins are made during the initial period of infection, whereas structural proteins of virus particles are produced subsequently Consequently, the mechanisms by which cellular and viral proteins cooperate to establish specific patterns of viral gene expression are the key to understanding progression through the infectious cycles of these viruses Properties of cellular RNA polymerases that transcribe viral DNA Eukaryotes have three transcriptional systems A general feature of eukaryotic cells is the division of transcriptional labor among three DNA dependent RNA polymerases RNA polymerase II makes precursors to both mRNAs and small, regulatory RNA molecules. The other two polymerases produce stable RNAs, such as ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) The evolution of RNA polymerases with distinct transcriptional responsibilities appears to be a device for maximizing opportunities for control of mRNA synthesis while maintaining a constant and abundant supply of the RNA species essential for the metabolism of all cells Despite their different functions, several of the 12 to 16 subunits of the large eukaryotic RNA polymerases are identical, while others are related in sequence to one another or to subunits of the bacterial RNA enzymes The structures of RNA polymerase II from various eukaryotes revealed that the organization of their active centers is similar to that of smaller DNA dependent RNA polymerases, as well as of enzymes that make DNA from DNA or RNA templates Transcription of cellular and viral genes requires not only template-directed synthesis of RNA but also correct interpretation of DNA punctuation signals that mark the sites at which transcription must start and stop Cellular RNA polymerases II and III transcribe viral templates Viral mRNAs or their precursors (pre-mRNAs) are made by RNA polymerase II in cells infected by many DNA viruses with both small and large genomes Some viral genomes must be converted to templates suitable for transcription Viral DNA molecules that are transcribed by cellular RNA polymerases must enter the infected cell nucleus, but there is considerable variation in the reactions needed to produce templates that can be recognized by the cellular machinery Viral DNA genomes that are partially or largely single-stranded, such as those of hepadnaviruses and parvoviruses, respectively, are converted to fully double-stranded DNA molecules by cellular enzymes Reverse transcription creates an appropriate double-stranded DNA template that includes the signals needed for its recognition by components of the cellular transcriptional machinery Many viral DNA genomes transcribed by RNA polymerase II are also organized by cellular nucleosomes, the fundamental structural units of the cellular chromatin templates transcribed by RNA polymerase II Because they are integrated into the cellular genome, the proviral DNA templates for retroviral transcription are organized into chromatin indistinguishable from that of the host cell Transcription by RNA polymerase II Accurate initiation of transcription by RNA polymerase II is directed by specific DNA sequences located near the initiation site, called the promoter Subsequently, the study of viral transcription yielded fundamental information about the mechanisms by which RNA polymerase II transcription is initiated and regulated The initiation reactions include promoter recognition, unwinding of the duplex DNA around the initiation site to form an open initiation complex, and movement of the transcribing complex away from the promoter Core promoters of viral and cellular genes contain all the information necessary for recognition of the site of initiation and assembly of precisely organized preinitiation complexes Short sequences, termed initiators, which specify accurate (but inefficient) initiation of transcription in the absence of any other promoter sequences, are also commonly found Regulation of RNA polymerase II transcription Numerous patterns of gene expression are necessary for eukaryotic life: some RNA polymerase II transcription units must be expressed in all cells, whereas others are transcribed only during specific developmental stages or in specialized differentiated cells Recognition of local and distant regulatory sequences Both local and distant sequences can control transcription from core promoters. However, local sequences are often sufficient for proper transcriptional regulation Efficient transcription of many viral and cellular genes also requires more distant regulatory sequences in the DNA template, which possess properties that were entirely unanticipated Enhancers are defined by their position and orientation-independent stimulation of transcription over distances as great as 10 kbp in the genome The simian virus 40 enhancer: a model for viral and cellular enhancers The simian virus 40 enhancer is built from three units, termed enhancer elements, that ensure transcription of the viral early gene and initiation of the viral infectious cycle in many different cellular environments Each enhancer element is subdivided into smaller sequence motifs recognized by DNAbinding proteins that are differentially produced in different cell types In contrast, some viral templates for RNA polymerase II transcription contain enhancers that are active only in a specific cell type, only in the presence of viral proteins, or only under particular metabolic conditions The simian virus 40 enhancer is located within 200 bp of the transcription initiation site, but enhancers of cellular genes are typically found thousands or tens of thousands of base pairs up or downstream of the promoters that they regulate Enhancers can, therefore, stimulate RNA polymerase II transcription by multiple molecular mechanisms The primary consequence of these mechanisms is to increase the probability that the gene(s) to which an enhancer 1s linked will be transcribed Common properties of proteins that regulate transcription The cellular and viral DNA binding proteins necessary for transcription from viral DNA templates share a number of common properties Their most characteristic feature is modular organization: they are built from discrete structural and functional domains Regulation of transcription by sequence-specific DNA binding proteins usually requires additional proteins termed coactivators or corepressors Coactivators can cooperate with multiple, sequence-specific activators and stimulate transcription from many promoters It is now clear that complicated patterns of histone posttranslational modification, particularly of the N-terminal tails of the four core histones of the nucleosome, govern the properties of chromatin Such histone acetyltransferases, and the deacetylases associated with corepressors, help establish the patterns of histone post-transcriptional modifications that distinguish transcriptionally active from inactive chromatin A second class of coactivators contain ATP dependent chromatin-remodeling enzymes that after the way in which DNA is bound to nucleosomes The proteins that control transcription are therefore frequently regulated by mechanisms that govern their activity, availability, or intracellular concentration These mechanisms include modulation of the phosphorylation (or other modifications) of specific amino acids, which can determine now well a protein binds to DNA, its oligomerization state, or the properties of its regulatory domain Transcription of viral DNA templates by the cellular machinery alone Although cellular proteins are sufficient for transcription of polyomavirus early genes, viral large T antigens are necessary for efficient late transcription In fact, the only three known examples of viral templates transcribed by cellular proteins alone are the double-stranded DNA genomes of caulimovirus of plants, and the integrated proviral DNAs In cells infected by many retroviruses The genomes of most retroviral species do not encode transcriptional regulators, so the rate at which proviral DNA is transcribed is determined by the constellation of cellular transcription proteins present in an infected cell Because the LTRs are direct repeats of one another, transcription directed by the 3' LTR extends into cellular DNA and cannot contribute to the expression of retroviral genetic information In fact, the transcriptional control region of the 3' LTR is normally inactivated by a process called promoter occlusion: the passage of transcribing complexes initiating at the 5' LTR through the 3' LTR prevents recognition of the latter by enhancer and promoter-binding proteins Absolute dependence on cellular components for the production of viral transcripts avoids the need to devote limited viral genetic capacity to transcriptional regulatory proteins Viral proteins that govern transcription of DNA templates Patterns of regulation Transcription of many viral DNA templates by the RNA polymerase II machinery results in the synthesis of large quantities of transcripts in relatively short periods The first [regulatory circuit] is a transcriptional cascade, in which different viral transcription units are activated in an ordered sequence, often accompanied by inhibition of transcription of genes expressed earlier This mechanism, which ensures that different classes of viral proteins are made during different periods of the infectious cycle, is characteristic of viruses with DNA genomes The second [regulatory circuit] is a positive autoregulatory loop, in which initial transcription and production of a viral protein leads to an increased rate of transcription of the same viral transcription unit The participation of viral proteins confers a measure of control lacking when the transcriptional program 1s executed solely by cellular components The human immunodeficiency virus type 1 Tat protein autoregulates transcription Like those of their simpler cousins, the proviruses of retroviruses with complex genomes contain a major transcription unit controlled by sequences in the 5' LTR, and in some cases encode additional (antisense) transcripts initiating within the 3' LTR The major transcription unit contains the coding sequences not only for the common structural proteins and enzymes but also for auxiliary proteins, including transcriptional regulators Cellular proteins initially direct transcription of the proviral DNA at some basal rate: among the processed products of the primary viral transcript are the spliced mRNAs from which the Tat protein is synthesized; this protein is imported into the nucleus, where it stimulates transcription of the proviral template upon binding to its RNA recognition site in nascent viral transcripts Cellular proteins recognize the HIV-1 LTR Cellular proteins that bind to the LTR enhancer and promoter proteins support a low rate of proviral transcription before Tat is made in infected cells Viral reproduction (i.e., transcription) in infected T cells in culture is stimulated by Tcell growth factors, indicating that viral transcription requires cellular components available only in such activated T cells Within the HIV-1 LTR, the promoter is immediately preceded by two important regulatory regions, termed the core and upstream enhancers, that are necessary for efficient viral transcription in both peripheral blood lymphocytes and certain T-cell lines Both the core and the upstream enhancers are densely packed with binding sites for cellular proteins, many of which are enriched in the types of cell In which the virus can reproduce Because these assays do not reproduce physiological conditions, a positive result establishes only that a certain protein can stimulate transcription, not that it normally does so Regulation of viral transcription by cellular pathways is exemplified by the critical role of the transcriptional activator NF-kB in replication of HIV-1 Growth factors that activate T cells trigger signal transduction cascades that lead to phosphorylation and subsequent degradation of these inhibitors by the cytoplasmic multiprotease complex (the proteasome) The severe, or complete, inhibition of virus reproduction (transcription) in human CD4+ T lymphocytes caused by mutations in the NF-kB binding sites emphasizes the importance of activation of this cellular protein in the infectious cycle The Tat protein regulates transcription by unique mechanisms Tat recognizes an RNA structure Stimulation of HIV-1 transcription by Tat requires an LTR sequence, termed the transactivation response (TAR) element, which lies within the transcription unit Indeed, the Tat protein binds specifically to a trinucleotide bulge and adjacent base pairs in the stem of the TAR RNA stem-loop structure Tat stimulates transcriptional elongation Binding of Tat to TAR RNA stimulates production of viral RNA by as much as 100-fold Complexes that initiate transcription in the absence of Tat elongate poorly, and many terminate transcription within 60 bp of the initiation site so that full-length transcripts of proviral DNA account for no more than 10% of the total How Tat stimulates transcriptional elongation A search for cellular proteins that stimulate viral transcription when bound to the Nterminal region of Tat identified the human Ser/Thr kinase P-TEFb (positive-acting transcription factor b), which was known to increase the efficiency of elongation of cellular transcripts Tat and P-TEFb bind cooperatively to the TAR RNA stem-loop (that is, with higher affinity than either protein alone), and with greater specificity The results of experiments in which P-TEFb was inhibited in infected cells, as well as genetic analyses, have established that P-TEFb is essential for Tat-dependent stimulation of viral transcription in vivo Association of the scaffolding subunit (AFF4) of this multi-protein complex with both Tat and cycling T1 recruits the complex to transcription elongation complexes stalled on proviral DNA These interactions stabilize flexible regions in all three proteins, increase the affinity of birding of cyclin T1 and Tat to the TAR RNA hairpin, and prevent degradation of normally short-lived subunits of the super-elongation complex Once associated with transcription complexes, the active kinase phosphorylates Ser residues within the C-terminal segment of the largest subunit of RNA polymerase II, which is hypophosphorylated when RNA polymerase II is present in preinitiation complexes, a modification that stimulates elongation Tat also facilitates nucleosome remodeling Although HIV-1 proviral DNA is integrated preferentially into or near transcriptionally active genes of the host cell, efficient transcription requires reorganization of nucleosomes, which are located at specific positions on this LTR In addition to binding to the cyclin T1 subunit of P-TEFb, Tat can bind to specific subunits of ATP dependent chromatin remodeling enzymes of the SWI/SNF family, as well as to several histone acetyltransferases What is the advantage of such unusual transcriptional regulation? However, without exception, these proteins and all known cellular transcriptional regulators operate via direct or indirect binding to specific DNA sequences Or it might be that the conformational flexibility of RNA allows for a degree of cooperative interactions and positive feedback among Tat and subunits of the superelongation complex that are less favorable when proteins bind to DNA sequences The transcriptional cascades of DNA viruses Common strategies are executed by virus-specific mechanisms Prior to initiation of genome replication, during early phases, infected cells synthesize viral proteins necessary for efficient viral gene expression, viral DNA synthesis, or other regulatory functions Transcription of the late genes, most of which encode structural proteins, requires genome replication. This property ensures coordinated production of the DNA genomes and the structural proteins from which progeny virus particles are assembled This type of organization reduces the genetic information that must be devoted to transcription punctuation marks and regulatory sequences, a significant advantage when genome size is limited by packaging constraints In fact, the potential for finely tuned regulation 1s much greater when the viral genome contains a large number of independent transcription units As might be anticipated, the sophistication of the regulatory circuits that govern the transcriptional cascade of these DNA viruses increases with genome size Synthesis of a single viral protein, LT, In simian virus 40 infected cells heads inevitably to entry into the late phase of infection: LT both induces initiation of viral DNA synthesis and activates late transcription Accumulation of progeny viral genomes leads to relief of repression of transcription of the gene that encodes the sequence-specific DNA birding protein IVa2 and subsequent activation of transcription from the major late promoter Examples of viral proteins that stimulate transcription These proteins all promote progression through the infectious cycle but differ in the mechanisms by which they become associated with viral promoters, and consequently in the specificity with which they stimulate transcription Some viral transcriptional regulators are closely similar to cellular proteins that bind to specific DNA sequences in promoters or enhancers These viral proteins possess discrete DNA binding domains, some with sequence motifs characteristic of cellular DNA birding proteins, and activation domains that interact with cellular initiation proteins The Epstein-Barr virus Zta protein: a sequence-specific DNA binding protein that induces entry into the production cycle Virus reproduction begins with synthesis of three viral proteins that regulate gene expression. However, just one of these, the transcriptional regulator Zta, is sufficient to interrupt latency and induce entry into the productive cycle The Zta protein exhibits many properties characteristic of the cellular proteins that recognize promoter sequences: it is a modular, sequence-specific DNA binding protein that belongs to the basic leucine zipper family The discrete activation domain, which can bind directly to cellular initiation proteins, such as submits of transcription factor IID, is thought to facilitate the assembly of preinitiation complexes, and hence initiation of transcription from these promoters Entry into the infectious cycle appears to be an inevitable consequence of production of active Zta: this protein not only stimulates transcription from the promoters of its own and other early genes but also plays an important role in replication from the lytic origins The herpes simplex virus type 1 VP16 protein: sequence-specific activation of transcription via a cellular DNA binding protein The VP16 protein lacks a DNA binding domain, but its acidic activation domain is one of the most potent known and has been exploited to investigate mechanisms of transcriptional activation When part of such fusion proteins, the VP16 acidic activation domain can bind to various coactivators, including the large, multiprotein assembly termed mediator, and stimulate several reactions required for initiation of transcription The 5' flanking regions of VP16 responsive viral immediate-early genes contain at least one copy of the consensus sequence that is necessary for VP16 dependent activation of their transcription, 5’TAATGARAT3’ An important function of HCFC1 appears to be stabilization of conformational change in VP16, to allow its high-affinity binding to OCT-1 on the immediate-early promoters The incorporation of the VP16 protein into virus particles at the end of one infectious cycle appears to be an effective way to ensure transcription of viral genes and initiation of viral reproduction in a new host cell Furthermore, it is a component of several chromatin-modifying complexes, and its recruitment to viral immediate-early promoters is required for replacement of repressive with activating modifications of the nucleosomal histones associated with these promoters Adenovirus E1A proteins: sequence-independent regulation of transcription by multiple mechanisms Two major E1A proteins are synthesized from differentially spliced mRNAs during the immediate-early phase of adenovirus infection As the larger E1A protein neither binds specifically to DNA nor depends on a specific promoter sequence, it is considered the prototypical example of viral proteins that stimulate transcription by indirect mechanisms The CR3 segment of the larger E1A protein comprises an N-terminal zinc finger motif followed by 10 amino acids that are highly conserved among human adenoviruses The short E1A CR3 sequence, like the activation domains of other viral transcriptional regulators we have discussed, stimulates multiple reactions in the transcription cycle Competition for RB by the E1A proteins disrupts the RB-E2F association and allows efficient transcription from E2F-dependent promoters, including that of the gene that encodes the proteins required for viral DNA synthesis Disruption of the interactions of these histone-modifying enzymes with specific activators by E1A proteins has been implicated In repression of enhancer-dependent transcription and is required for induction of cell proliferation Coordination of transcription of late genes with viral DNA synthesis In cells infected by the DNA viruses, synthesis of the large quantities of structural proteins needed for assembly of progeny virus particles is restricted to the late phase of infection, following the onset of viral genome replication The restriction of synthesis of structural proteins to the end of an infectious cycle of viral reproduction results from the dependence of late gene transcription on viral DNA replication: drugs or mutations that inhibit viral DNA synthesis in infected cells block efficient expression of late genes Titration of cellular repressors The most obvious consequence of viral DNA genome replication is the large increase in concentration of viral DNA molecules As soon as viral DNA synthesis begins, this number increases rapidly to values as high as hundreds of thousands of viral DNA molecules per infected cell nucleus Viral DNA replication increases the concentration of the late promoter until it exceeds that of repressor, and therefore allows this promoter to become active The IVa2 protein cooperates with a second viral protein (an L4 protein) that also binds to specific DNA sequences to stimulate the rate of initiation of transcription from the major late promoter, which controls production of structural proteins Activation of late promoters is therefore coupled indirectly to adenovirus DNA synthesis: this process initiates a transcriptional cascade in which late promoters are activated sequentially Although viral DNA synthesis is sufficient for activation of transcription of some viral late genes (e.g., the adenoviral IVa2 gene), this process is usually facilitated by one or more viral proteins Coupling to initial rounds of replication However, analysis of proteins associated with unreplicated viral genomes and viral replication forks supports a model in which formation of replication forks allows recruitment of cellular proteins that induce promoter escape Regulation of transcription termination Transcription of viral late genes is generally regulated by inhibition or activation of early reactions, like initiation or promoter clearance During the early phase of infection, major late transcription terminates within a region in the middle of the transcription unit Viral DNA synthesis is necessary to induce full-length transcription to a termination site close to the right-hand end of the viral genome, and expression of the many other major late coding sequences Availability and structure of templates Newly replicated viral DNA molecules can enter into additional replication cycles, serve as templates for transcription, or become assembled into virus particles, with different fates predominating at different times in the infectious cycle Although transcription of all viral DNA molecules made in infected cells would seem to be a simple mechanism to ensure efficient transcription of late genes, no more than 5 to 10% of the large numbers that accumulate are transcriptionally active Entry into one of two alternative transcriptional programs As in bacteriophage lambda lysogeny, latent infections are characterized by activation of a unique, latent phase transcriptional program characterized by expression of a very small set of viral genes In the latent state, transcription of lytic genes is blocked and only a single transcription unit is expressed efficiently as latency-associated transcripts (LATs) In latently infected neurons, lytic genes are organized by nucleosomes that carry repressive posttranslational modifications and are associated with cellular repressors of transcription This difference depends on binding sites within the LAT transcription unit for proteins that maintain boundaries between active and repressed chromatin: their deletion results in repressive modifications of LAT DNA associated nucleosomes The major 2.0 kb LATs, which accumulate to 40,000 to 100,000 copies in nuclei of latently infected neurons, lack poly(A) tails and are not linear molecules The first [stage], which can be induced by signaling pathways downstream of nerve growth factor or stress, comprises a transient wave of expression of all viral lytic genes, independently of VP16 synthesis or viral genome replication The second phase, entry into the lytic transcriptional program, does require VP16dependent stimulation of transcription Transcription of viral genes by RNA polymerase III The VA-RNA I genre specifies an RNA that counters the effects of a host cell defense mechanism and also serves as a precursor for production of viral miRNAs The VA-RNA I promoter Transcription of this gene, like all recognized by RNA polymerase III, depends on two intragenic sequences, the A and B boxes The internal sequences are recognized by the RNA polymerase III-specific initiation protein TFIIIC, which binds to the promoter to seed assembly of an initiation complex that also contains TFIIIB and the enzyme Inhibition of the cellular transcriptional machinery Cellular resources, such as substrates for RNA synthesis, can be devoted exclusively to the production of viral mRNAs (and, in many cases, RNA genomes), and competition between viral and cellular mRNAs for components of the translational machinery is minimized Selective transcription of viral genes is accompanied by loss of RNA polymerase II phosphorylated at a specific amino acid, induced by the viral ICP22 protein, and proteasomal degradation of this hypophosphorylated form of the enzyme correlates with inhibition of transcription of cellular genes Reproduction of the majority of viruses with RNA genomes requires neither the cellular transcriptional machinery nor its RNA products and is often accompanied by inhibition of cellular mRNA synthesis