Multisubunit DNA-dependent RNA Polymerase Research Articles

Genistein is effective in inhibiting Orf virus infection in vitro by targeting viral RNA polymerase subunit RPO30 protein.

Orf virus (ORFV), a typical member of the genus Parapoxvirus, Poxvirus family, causes a contagious pustular dermatitis in sheep, goats, and humans. Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm, which is a viral factor essential to poxvirus replication. Due to its vital role in viral life, vRNAP has emerged as one of the potential drug targets. In the present study, we investigated the antiviral effect of genistein against ORFV infection. We provided evidence that genistein exerted antiviral effect through blocking viral genome DNA transcription/replication and viral protein synthesis and reducing viral progeny, which were dosedependently decreased in genistein-treated cells. Furthermore, we identified that genistein interacted with the vRNAP RPO30 protein by CETSA, molecular modeling and Fluorescence quenching, a novel antiviral target for ORFV. By blocking vRNAP RPO30 protein using antibody against RPO30, we confirmed that the inhibitory effect exerted by genistein against ORFV infection is mediated through the interaction with RPO30. In conclusion, we demonstrate that genistein effectively inhibits ORFV transcription in host cells by targeting vRNAP RPO30, which might be a promising drug candidate against poxvirus infection.

Structural Studies of Eukaryotic RNA Polymerase I Using Cryo-Electron Microscopy.

Technical advances have pushed the resolution limit of single-particle cryo-electron microscopy (cryo-EM) throughout the past decade and made the technique accessible to a wide range of samples. Among them, multisubunit DNA-dependent RNA polymerases (Pols) are a prominent example. This review aims at briefly summarizing the architecture and structural adaptations of Pol I, highlighting the importance of cryo-electron microscopy in determining the structures of transcription complexes.

An Insight into the Proofreading Functions of Multisubunit DNA-Dependent RNA Polymerases and Their Catalytic Mechanism

Aim: To analyze the active sites of the proofreading (PR) functions in the multisubunit DNA-dependent RNA polymerases (MSU RNAPs) from prokaryotes, chloroplasts and eukaryotes, and propose a plausible unified catalytic mechanism for these enzymes.
 Study Design: Data collected on these enzymes from bioinformatics, biochemical, site-directed mutagenesis (SDM), X-ray crystallography and cryo-electron microscopy (cryo-EM) were used for the analyses.
 Methodology: The protein sequence data of MSU RNAPs from prokaryotes, prokaryotic-types (plant chloroplasts) and eukaryotes were obtained from PUBMED and SWISS-PROT databases. The advanced version of Clustal Omega was used for protein sequence analysis. Along with the conserved motifs identified by the bioinformatics analysis, the data already available from biochemical and SDM experiments, and X-ray crystallographic and cryo-EM data on these enzymes are also used to confirm the possible amino acids involved in the active site of the PR function in these MSU RNAPs
 Results: All the seven types of MSU RNAPs (I-VII) reported from prokaryotes to eukaryotes were analyzed by the multiple sequence alignment (MSA) software, Clustal Omega, to find out conservations among them. The MSA analysis showed many conserved amino acid motifs including small and large peptide regions from the MSU RNAPs of prokaryotes, eukaryotes and plant chloroplasts. Interestingly, the catalytic amino acid and template-binding pairs are highly conserved in all these polymerases, with a few exceptions. Most of them use a basic amino acid (R/K/H) for initiating catalysis and an -YG/FG- pair for template-binding. Some odd type of catalytic amino acids and template-binding pairs are observed in human pathogens, parasites and organisms which cannot ferment sugars. In all the MSU RNAPs, the proposed polymerase catalytic region also possessed three invariant Cs and an invariant H within it. The invariant Cs is shown to bind a zinc atom and proposed to involve in the PR function by excising any misincorporated nucleotide during the transcription process. In the plant-specific MSU RNAPs IV and V, which involve in transcriptional gene silencing in plants, the catalytic and template-binding pairs do not follow the regular distance conservations as observed with other five of the MSU RNAPs. Their polymerase/PR active site regions are similar to RNAP III rather than to RNAP II, as all three make only low molecular weight RNAs.
 Conclusions: All the known MSU RNAPs possess three invariant Cs and an invariant H embedded within the polymerase active site itself. The three invariant Cs are shown to bind a zinc atom and the invariant H could act as the proton acceptor from a metal-bound water molecule, for initiating excision of the mismatches by a Zn-mediated hydrolysis. Thus, the PR function in MSU RNAPs is integrated within the polymerase active site itself, which is in sharp contrast to the PR functions reported in DNA-dependent DNA polymerases and RNA-dependent RNA polymerases. Therefore, all the seven MSU RNAPs from prokaryotes and eukaryotes are proposed to follow a unified mechanism to excise the mismatches during transcription. The discovery of intrinsic self-correcting RNA transcription mechanism fulfils the missing link in molecular evolution.

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes

Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNAGln. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen etal., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.

Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Aim: To analyze the most complex multi-subunit (MSU) DNA dependent RNA polymerases (RNAPs) of eukaryotic organisms and find out conserved motifs, metal binding sites and catalytic regions and propose a plausible mechanism of action for these complex eukaryotic MSU RNAPs, using yeast (Saccharomyces cerevisiae) RNAP II, as a model enzyme.
 Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis and X-ray crystallographic data were analyzed.
 Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007- 2013.
 Methodology: Bioinformatics, Biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data of the enzyme were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNAPs from various eukaryotic sources. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical and SDM experiments and X-ray crystallographic analysis of these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis.
 Results: Multiple sequence alignment (MSA) of RNAPs from different eukaryotic organisms showed a large number of highly conserved motifs among them. Possible catalytic regions in the catalytic subunits of the yeast Rpb2 (= β in eubacteria) and Rpb1 (= β’ in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that was reported for DNA polymerases and single subunit (SSU) RNAPs. However, the invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU RNAPs, prokaryotic MSU RNAPs and DNA polymerases is also highly conserved in eukaryotic Rpb2 initiation subunits, but unusually a KG pair is found in higher eukaryotes including the human RNAPs. Like the eubacterial initiation subunits of MSU RNAPs, the eukaryotic initiation subunits, viz. Rpb2, exhibit very similar active site and catalytic regions but slightly different distance conservations between the template binding YG/KG pair and the catalytic R. In the eukaryotic initiation subunits, the proposed catalytic R is placed at the -9th position from the YG/KG pair and an invariant R is placed at -5 which are implicated to play a role in nucleoside triphosphate (NTP) selection as reported for SSU RNAPs (viral family) and DNA polymerases.
 Similarly, the eukaryotic elongation subunits (Rpb1) are also found to be very much homologous to the elongation subunits (β’) of prokaryotes. Interestingly, the catalytic regions are highly conserved, and the metal binding sites are absolutely conserved as in prokaryotic MSU RNAPs. In eukaryotes, the template binding YG pair is replaced with an FG pair. Another interesting observation is, similar to the prokaryotic β’ subunits, in the eukaryotic Rpb1 elongation subunits also, the proposed catalytic R is placed double the distance, i.e., -18 amino acids downstream from the FG pair unlike in the SSU RNAPs and DNA polymerases where the distance is only -8 amino acids downstream from the YG pair. Thus, the completely conserved FG pair, catalytic R with an invariant R, at -6th position are proposed to play a crucial role in template binding, NTP selection and polymerization reactions in the elongation subunits of eukaryotic MSU RNAPs. Moreover, the Zn binding motif with the three completely conserved Cs is also highly conserved in the eukaryotic elongation subunits. Another important difference is that the catalytic region is placed very close to the N-terminal region in eukaryotes.
 Conclusions: Unlike reported for the DNA polymerases and SSU RNA polymerases, the of eukaryotic MSU RNAPs use an R as the catalytic amino acid and exhibit a different distance conservation in the initiation and elongation subunits. An invariant Zn2+ binding motif found in the Rpb1 elongation subunits is proposed to participate in proof-reading function. Differences in the active sites of bacterial and human RNA polymerases may pave the way for the design of new and effective drugs for many bacterial infections, including the multidrug resistant strains which are a global crisis at present.

The study of the phiKZ phage non-canonical non-virion RNA polymerase

Non-canonical multisubunit DNA-dependent RNA-polymerases (RNAP) form a new group of the main transcription enzymes, which have only distinct homology to the catalytic subunits of canonical RNAPs of bacteria, archaea and eukaryotes. One of the rare non-canonical RNAP, which was partially biochemically characterized, is non-virion RNAP (nvRNAP) encoded by Pseudomonas phage phiKZ. PhiKZ nvRNAP consists of five subunits, four of which are homologs of β and β’ subunit of bacterial RNAP, and the fifth subunits with unknown function. To understand the role of the fifth subunit in phiKZ nvRNAP, we created co-expression system allowing to get recombinant full five-subunit (5s) and four-subunit (4s) complexes and performed their comparison. The 5s recombinant complex is active on phage promoters in vitro as the native nvRNAP. The 4s complex cannot extend RNA, so 4s complex is not a catalytically active core of phiKZ nvRNAP. Thus, the phiKZ fifth subunit is not only a promoter-recognition subunit, but it plays an important role in the formation of active phiKZ nvRNAP.

"Multi-subunit RNA Polymerases of Bacteria: An insight into their Active Sites and Catalytic Mechanism"

Objectives: To analyze the Multi-Subunit (MSU) DNA dependent RNA polymerases of eubacteria and identify conserved motifs, active site regions among them and propose a plausible mechanism of action for these enzymes, using the E. coli RNA polymerase as a model system. Methods: The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNA polymerases from eubacteria. Along with the conserved motifs identified by the bioinformatics analysis, the data already available from biochemical and Site-Directed Mutagenesis (SDM) experiments and X-ray crystallographic analysis on these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis. Findings: Multiple Sequence Alignment (MSA) of eubacterial RNA polymerases from different bacterial sources showed only a very few highly conserved motifs among them. Possible catalytic regions in the catalytic subunits β and β‘ (in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that is reported for DNA polymerases and single subunit (SSU)DNA dependent RNA polymerases. In addition to, the invariant ‘gatekeeper/DNA template binding’ YG pair is also found in all the MSU RNA polymerases as found in all the SSU RNA polymerases and DNA polymerases. The eubacterial βsubunits are very similar in theiractive sites, catalytic regions and distance conservations between the template binding YG pair and the catalytic R, i.e., they are placed at 7 amino acids length. An invariant R is placed here at -6th position from the catalytic R, similar to SSU RNA polymerases and DNA polymerases which is proposed to play an important role in NTP selection. In the eubacterial β’ catalytic subunits, the proposed catalytic R is placed double the distance, i.e., -16 amino acids downstream from the YG pair unlike in the SSU RNA polymerasesand DNA polymerases where the distance is only 8th amino acids downstream from the YG pair. The two Ds in the completely conserved metal binding region could act as the charge shielder and orient the α-phosphates of incoming NTPs for reaction at the 3’-OH growing end. Based on the sequence analysis, a model is proposed for the initiation and elongation steps in the transcription process and a plausible mechanism of action. Applications: A comparative analysis of the bacterial and human RNA polymerases for their transcription mechanism will pave way for design new and effective drugs for many bacterial infections, including the antibiotic resistance. Keywords: Bacterial RNA Polymerases, Clustal Omega, Conserved Motifs, Multi-Subunit RNA Polymerases, Polymerase Active Sites, Polymerization Mechanism, Proofreading

Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure.

The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size.

In Vitro Transcription Assays and Their Application in Drug Discovery.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein

The multi-subunit DNA-dependent RNA polymerase (RNAP) is the principal enzyme of transcription for gene expression. Transcription is regulated by various transcription factors. Gre factor homologue 1 (Gfh1), found in the Thermus genus, is a close homologue of the well-conserved bacterial transcription factor GreA, and inhibits transcription initiation and elongation by binding directly to RNAP. The structural basis of transcription inhibition by Gfh1 has remained elusive, although the crystal structures of RNAP and Gfh1 have been determined separately. Here we report the crystal structure of Thermus thermophilus RNAP complexed with Gfh1. The amino-terminal coiled-coil domain of Gfh1 fully occludes the channel formed between the two central modules of RNAP; this channel would normally be used for nucleotide triphosphate (NTP) entry into the catalytic site. Furthermore, the tip of the coiled-coil domain occupies the NTP β-γ phosphate-binding site. The NTP-entry channel is expanded, because the central modules are 'ratcheted' relative to each other by ∼7°, as compared with the previously reported elongation complexes. This 'ratcheted state' is an alternative structural state, defined by a newly acquired contact between the central modules. Therefore, the shape of Gfh1 is appropriate to maintain RNAP in the ratcheted state. Simultaneously, the ratcheting expands the nucleic-acid-binding channel, and kinks the bridge helix, which connects the central modules. Taken together, the present results reveal that Gfh1 inhibits transcription by preventing NTP binding and freezing RNAP in the alternative structural state. The ratcheted state might also be associated with other aspects of transcription, such as RNAP translocation and transcription termination.

Deletion of Switch 3 Results in an Archaeal RNA Polymerase That Is Defective in Transcript Elongation

Switch 3 is a polypeptide loop conserved in all multisubunit DNA-dependent RNA polymerases (RNAPs) that extends into the main cleft of the RNAP and contacts each base in a nascent transcript as that base is released from the internal DNA-RNA hybrid. Plasmids have been constructed and transformed into Thermococcus kodakaraensis, which direct the constitutive synthesis of the archaeal RNAP subunit RpoB with an N-terminal His(6) tag and the Switch 3 loop either intact (wild-type) or deleted (DeltaS3). RNAPs containing these plasmid-encoded RpoB subunits were purified, and, in vitro, the absence of Switch 3 had no negative effects on transcription initiation or elongation complex stability but reduced the rate of transcript elongation. The defect in elongation occurred at every template position and increased the sensitivity of the archaeal RNAP to intrinsic termination. Comparing these properties and those reported for a bacterial RNAP lacking Switch 3 argues that this loop functions differently in the RNAPs from the two prokaryotic domains. The close structural homology of archaeal and eukaryotic RNAPs would predict that eukaryotic Switch 3 loops likely conform to the archaeal rather than bacterial functional paradigm.

Evolutionary History of Plant Multisubunit RNA Polymerases IV and V: Subunit Origins via Genome-Wide and Segmental Gene Duplications, Retrotransposition, and Lineage-Specific Subfunctionalization

Eukaryotes have three multisubunit DNA-dependent RNA polymerases that are essential for viability, abbreviated as Pol I, Pol II, and Pol III. Remarkably, Arabidopsis thaliana and other higher plants contain two additional nuclear multisubunit RNA polymerases, Pol IV and Pol V. These plant-specific polymerases are not essential for viability but have nonredundant roles in RNA-mediated gene-silencing pathways. Proteomic analyses have revealed that Arabidopsis Pol IV and Pol V have a 12-subunit composition like Pol II. In fact, half of the subunits of Pols II, IV, and V are encoded by the same genes. The remaining Pol IV- or Pol V-specific subunit genes arose through duplication and subfunctionalization of ancestral Pol II subunit genes. These include the genes encoding the largest subunits unique to Pol IV or Pol V, the genes encoding the second- and the fourth-largest subunits that are used by both Pol IV and Pol V, the gene encoding the fifth-largest subunit unique to Pol V and the genes encoding the seventh-largest subunits that are unique to Pol IV and Pol V. On the basis of phylogenetic reconstructions, the gene duplication events giving rise to the first-, second-, fourth-, fifth-, and seventh-largest subunits of Pol IV and/or Pol V occurred independently. Interestingly, a cDNA-mediated duplication of the Pol II seventh-largest subunit gene via retro-tranposition was an early event in Pol IV evolution, preceded only by the duplications of the largest and second-largest subunit genes. Secondary duplication of this cDNA-like gene to generate Pol IV- and Pol V-specific seventh-largest subunits has occurred in Arabidopsis but not all dicotyledonous plants or monocots, indicative of the dynamic evolution of RNA Pol IV and Pol V in plants.

Molecular Evolution of Multisubunit RNA Polymerases: Structural Analysis

Comprehensive multiple sequence alignments of the multisubunit DNA-dependent RNA polymerase (RNAP) large subunits, including the bacterial β and β′ subunits and their homologs from archaebacterial RNAPs, eukaryotic RNAPs I–III, nuclear–cytoplasmic large double-stranded DNA virus RNAPs, and plant plastid RNAPs, were created [Lane, W. J. and Darst, S. A. (2009). Molecular evolution of multisubunit RNA polymerases: sequence analysis. In press]. The alignments were used to delineate sequence regions shared among all classes of multisubunit RNAPs, defining common, fundamental RNAP features as well as identifying highly conserved positions. Here, we present a systematic, detailed structural analysis of these shared regions and highly conserved positions in terms of the RNAP structure, as well as the RNAP structure/function relationship, when known.

A zinc-binding site in the largest subunit of DNA-dependent RNA polymerase is involved in enzyme assembly.

All multisubunit DNA-dependent RNA polymerases (RNAP) are zinc metalloenzymes, and at least two zinc atoms are present per enzyme molecule. RNAP residues involved in zinc binding and the functional role of zinc ions in the transcription mechanism or RNAP structure are unknown. Here, we locate four cysteine residues in the Escherichia coli RNAP largest subunit, beta', that coordinate one of the two zinc ions tightly associated with the enzyme. In the absence of zinc, or when zinc binding is prevented by mutation, the in vitro-assembled RNAP retains the proper subunit stoichiometry but is not functional. We demonstrate that zinc acts as a molecular chaperone, converting denatured beta' into a compact conformation that productively associates with other RNAP subunits. The beta' residues coordinating zinc are conserved throughout eubacteria and chloroplasts, but are absent from homologs from eukaryotes and archaea. Thus, the involvement of zinc in the RNAP assembly may be a unique feature of eubacterial-type enzymes.

A new vaccinia virus intermediate transcription factor.

Transcription of the vaccinia virus genome is mediated by a virus-encoded multisubunit DNA-dependent RNA polymerase in conjunction with early-, intermediate-, and late-stage-specific factors. Previous studies indicated that two virus-encoded proteins (capping enzyme and VITF-1) and one unidentified cellular protein (VITF-2) are required for specific transcription of an intermediate promoter template in vitro. We have now extensively purified an additional virus-induced intermediate transcription factor with a native mass of approximately 100 kDa.

A cellular factor is required for transcription of vaccinia viral intermediate-stage genes.

The cytoplasmic location of vaccinia virus replication and evidence that the multisubunit DNA-dependent RNA polymerase, early and late stage transcription factors, capping and methylating enzymes, and poly(A) polymerase are virus encoded raised the possibility that all of the proteins needed for viral mRNA synthesis are of viral origin. Previous studies showed that four components from infected cells, the viral RNA polymerase and capping enzyme and two factors called vaccinia virus intermediate transcription factors (VITFs) 1 and 2, can reconstitute transcription of vaccinia virus intermediate-stage genes in vitro. Here, we demonstrate that VITF-2 can be isolated from the nuclei of uninfected HeLa cells as well as from the cytoplasm of infected cells. The proteins with VITF-2 activity from uninfected and infected cells cochromatographed and cosedimented, suggesting that they are identical. VITF-2 activity was found in extracts of other uninfected human and monkey cells but not in nonpermissive Trichoplusia ni insect cells or in conditionally permissive rabbit kidney 13 cells. VITF-2 activity was present, however, in a permissive line of rabbit kidney 13 cells that had been stably transfected with the vaccinia virus K1L host range gene. We suggest that the VITF-2 level acts as a gauge of the permissive state of the cell and thereby regulates the length of the early prereplicative phase of the infection.

RNA polymerase-associated protein Rap94 confers promoter specificity for initiating transcription of vaccinia virus early stage genes.

The association of a 94,000-Da viral polypeptide, called Rap94, with 30-40% of the multisubunit DNA-dependent RNA polymerase molecules purified from infectious vaccinia virus particles was established by immunoaffinity chromatography. The submolar amount of Rap94, relative to RNA polymerase, was confirmed by quantitative immunoblotting of total virion extracts. Only the RNA polymerase molecules containing Rap94 could functionally interact with VETF, the vaccinia virus early transcription factor, to transcribe a double-stranded DNA template regulated by a viral early stage promoter. Rap94 was required for the synthesis of short oligoribonucleotides and for the formation of stable ternary transcription complexes. With a nonspecific single-stranded DNA template, however, the Rap94-deficient polymerase had greater catalytic activity than the Rap94-containing polymerase. These data support a model in which Rap94 confers specificity to the RNA polymerase for promoters of early stage genes.

DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.