Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Influenza virus RNA polymerase is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the viral RNA genome. The catalytic site for RNA polymerization is located on the PB1 subunit. To identify the primer ATP-binding site, we have employed a highly selective cross-linking technique: three structurally diverse ATP analogues with reactive groups on their phosphate moieties were first cross-linked to the viral RNA polymerase, and the cross-linked nucleotides were then elongated to dinucleotides by adding the second substrate [alpha-(32)P]GTP. Only the ATP analogues tethered to the primer-binding site could be elongated to radioactive AG dinucleotides. Using this catalytically competent cross-linking procedure, the PB1 subunit was found to be the only labeled subunit. Limited proteolysis of the labeled PB1 by V8 protease revealed the segment between amino acids 179 and 297 as the only bearer of the radioactive label. Thus, we concluded that this region of PB1 faces the 5' end of the primer nucleotide. In support of this prediction, the cross-linked dinucleotides were further elongated up to 8-10 nucleotides in length upon addition of all four substrates. This result suggests that the substantial mass of RNA can be accommodated between the binding site for the primer (and nascent RNA) and the catalytic center of RNA polymerization.
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PMID:Structure-function relationship of the influenza virus RNA polymerase: primer-binding site on the PB1 subunit. 1513 62

The phoPR operon encodes a response regulator, PhoP, and a histidine kinase, PhoR, which activate or repress genes of the Bacillus subtilis Pho regulon in response to an extracellular phosphate deficiency. Induction of phoPR upon phosphate starvation required activity of both PhoP and PhoR, suggesting autoregulation of the operon, a suggestion that is supported here by PhoP footprinting on the phoPR promoter. Primer extension analyses, using RNA from JH642 or isogenic sigE or sigB mutants isolated at different stages of growth and/or under different growth conditions, suggested that expression of the phoPR operon represents the sum of five promoters, each responding to a specific growth phase and environmental controls. The temporal expression of the phoPR promoters was investigated using in vitro transcription assays with RNA polymerase holoenzyme isolated at different stages of Pho induction, from JH642 or isogenic sigE or sigB mutants. In vitro transcription studies using reconstituted EsigmaA, EsigmaB, and EsigmaE holoenzymes identified PA4 and PA3 as EsigmaA promoters and PE2 as an EsigmaE promoter. Phosphorylated PhoP (PhoP approximately P) enhanced transcription from each of these promoters. EsigmaB was sufficient for in vitro transcription of the PB1 promoter. P5 was active only in a sigB mutant strain. These studies are the first to report a role for PhoP approximately P in activation of promoters that also have activity in the absence of Pho regulon induction and an activation role for PhoP approximately P at an EsigmaE promoter. Information concerning PB1 and P5 creates a basis for further exploration of the regulatory coordination or overlap of the PhoPR and SigB regulons during phosphate starvation.
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PMID:Autoinduction of Bacillus subtilis phoPR operon transcription results from enhanced transcription from EsigmaA- and EsigmaE-responsive promoters by phosphorylated PhoP. 1520 29

The RNA-dependent RNA polymerase of influenza virus consists of three subunits, PB1, PB2, and PA, and synthesizes three kinds of viral RNAs, vRNA, cRNA, and mRNA. PB1 is a catalytic subunit; PB2 recognizes the cap structure for generation of the primer for transcription; and PA is thought to be involved in viral RNA replication. However, the process of polymerase complex assembly and the exact nature of polymerase complexes involved in synthesis of the three different RNA species are not yet clear. ts53 virus is a temperature-sensitive (ts) mutant derived from A/WSN/33 (A. Sugiura, M. Ueda, K. Tobita, and C. Enomoto, Virology 65:363-373, 1975). We confirmed that the mRNA synthesis level of ts53 remains unaffected at the nonpermissive temperature, whereas vRNA synthesis is largely reduced. Sequencing of the gene encoding ts53 PA and recombinant virus rescue experiments revealed that an amino acid change from Leu to Pro at amino acid position 226 is causative of temperature sensitivity. By glycerol density gradient analyses of nuclear extracts prepared from wild-type virus-infected cells, we found that polymerase proteins sediment in three fractions: one (H fraction) consists of RNP complexes, another (M fraction) contains active polymerases but not viral RNA, and the other (L fraction) contains inactive forms of polymerases. Pulse-chase experiments showed that polymerases in the L fraction are converted to those in the M fraction. In ts53-infected cells, polymerases accumulated in the L fraction. These results strongly suggest that PA is involved in the assembly of functional viral RNA polymerase complexes from their inactive intermediates.
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PMID:Involvement of influenza virus PA subunit in assembly of functional RNA polymerase complexes. 1561 1

Influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex of PB1, PB2, and PA. We show that the individually expressed PB2 subunit can be assembled with the coexpressed PB1-PA dimer in vitro into a transcriptionally active complex. Furthermore, we demonstrate that a model viral RNA promoter can bind to the PB1-PA dimer prior to assembly with PB2. Our results are consistent with a recently proposed model for the sequential assembly of viral RNA polymerase complex in which the PB1-PA dimeric complex and the PB2 monomer are transported into the nucleus separately and then assembled in the nucleus.
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PMID:In vitro assembly of PB2 with a PB1-PA dimer supports a new model of assembly of influenza A virus polymerase subunits into a functional trimeric complex. 1595 11

Influenza virus RNA polymerase is a heterotrimeric complex consisting of PB1, PB2, and PA subunits. These polymerase subunits accumulate in the nucleus of infected cells. We report here that PB2, from both human and avian influenza viruses, could also localize to mitochondria in transfected cells. Importantly, cells infected with influenza A virus also displayed mitochondrial PB2. We show that an N-terminal motif composed of 120 amino acids is sufficient for localization of PB2 to mitochondria. In particular, leucine residues at positions 7 and 10 were essential for mitochondrial targeting. Recombinant influenza A/WSN/33 viruses expressing PB2 proteins with L7A and/or L10A mutations showed reduced viral titers, but unaffected levels of transcription, replication, and protein expression. The introduction of L7A and/or L10A mutations into recombinant viruses correlated with reduced mitochondrial membrane potential in infected cells, suggesting that mitochondrial localization of PB2 contributes to the preservation of mitochondrial function during influenza virus infection.
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PMID:Characterization of a mitochondrial-targeting signal in the PB2 protein of influenza viruses. 1624 67

The influenza A virus RNA-dependent RNA polymerase consists of three subunits PB1, PB2 and PA. The 5' and 3' terminal sequences of the viral RNA (vRNA) form the viral promoter and are bound by the PB1 subunit. The putative promoter-binding sites of the PB1 subunit have been mapped in previous studies but with contradictory results. The aim of the current study was to investigate the function of two evolutionary conserved regions in PB1 - from aa 233 to 249 and 269 to 281, which lie immediately N- and C-terminal, respectively, of a previously proposed binding site for the 3' end of the vRNA promoter. The previously proposed binding site extended from aa 249 to 256 and centred on two phenylalanine residues (F251 and F254). However, the fact that F251 is required for polymerase activity was not confirmed here. Instead, it was proposed that the 233-249 region contains a new 5' vRNA promoter-binding site, and arginine residues crucial for this activity were characterized. However, residues 269-281 were unlikely to be directly involved in promoter binding. These results are discussed in relation to the previous studies and a new model for vRNA promoter binding to the influenza RNA polymerase is presented.
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PMID:A new promoter-binding site in the PB1 subunit of the influenza A virus polymerase. 1647 91

Influenza viruses replicate and transcribe their segmented negative-sense single-stranded RNA genome in the nucleus of the infected host cell. All RNA synthesising activities associated with influenza virus are performed by the virally encoded RNA-dependent RNA polymerase (RdRp) that consists of three subunits, PA, PB1 and PB2. However, viral transcription is critically dependent on on-going cellular transcription, in particular, on activities associated with the cellular DNA-dependent RNA polymerase II (Pol II). Thus, the viral RdRp uses short 5' capped RNA fragments, derived from cellular Pol II transcripts, as primers for viral mRNA synthesis. These capped RNA primers are generated by cleavage of host Pol II transcripts by an endonuclease activity associated with the viral RdRp. Moreover, some viral transcripts require splicing and since influenza virus does not encode splicing machinery, it is dependent on host splicing, an activity also related to Pol II transcription. Despite these functional links between viral and host Pol II transcription, there has been no evidence that a physical association existed between the two transcriptional machineries. However, recently it was reported that there is a physical interaction between the trimeric viral RdRp and cellular Pol II. The viral RdRp was found to interact with the C-terminal domain (CTD) of initiating Pol II, at a stage in the transcription cycle when capping takes place. It was therefore proposed that this interaction may be required for the viral RNA (vRNA) polymerase to gain access to capped RNA substrates for endonucleolytic cleavage. The virus not only relies on cellular factors to support its own RNA synthesis, but also subverts cellular pathways in order to generate an environment optimised for viral multiplication. In this respect, the interaction of the viral NS1 protein with factors involved in cellular pre-mRNA processing is of particular relevance. The virus also alters the distribution of Pol II on cellular genes, leading to a reduction in elongating Pol II thereby contributing to the phenomenon known as host shut-off.
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PMID:Functional association between viral and cellular transcription during influenza virus infection. 1693 65

Both transcription and replication of the influenza virus RNA genome are catalysed by a virus-specific RNA polymerase. Recently, an in vitro assay, based on the synthesis of pppApG, for the initiation of replication by recombinant RNA polymerase in the absence of added primer was described. Here, these findings are extended to show that adenosine, AMP and ADP can each substitute for ATP in reactions catalysed by either recombinant ribonucleoprotein or RNA polymerase complexes with either model virion RNA (vRNA) or cRNA promoters. The use of either adenosine or AMP, rather than ATP, provides a convenient, sensitive and easy assay of replication initiation. Moreover, no pppApG was detected when a PB1-PA dimer, rather than the trimeric polymerase, was used to catalyse synthesis, contrasting with a previous report using baculovirus-expressed influenza RNA polymerase. Overall, it is suggested that the heterotrimeric polymerase is essential for the initiation of replication.
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PMID:Role of the influenza virus heterotrimeric RNA polymerase complex in the initiation of replication. 1703 Aug 72

Transcription and replication of the influenza virus RNA genome occur in the nuclei of infected cells through the viral RNA-dependent RNA polymerase consisting of PB1, PB2, and PA. We previously identified a host factor designated RAF-1 (RNA polymerase activating factor 1) that stimulates viral RNA synthesis. RAF-1 is found to be identical to Hsp90. Here, we examined the intracellular localization of Hsp90 and viral RNA polymerase subunits and their molecular interaction. Hsp90 was found to interact with PB2 and PB1, and it was relocalized to the nucleus upon viral infection. We found that the nuclear transport of Hsp90 occurs in cells expressing PB2 alone. The nuclear transport of Hsp90 was in parallel with that of the viral RNA polymerase binary complexes, either PB1 and PB2 or PB1 and PA, as well as with that of PB2 alone. Hsp90 also interacted with the binary RNA polymerase complex PB1-PB2, and it was dissociated from the PB1-PB2 complex upon its association with PA. Furthermore, Hsp90 could form a stable PB1-PB2-Hsp90 complex prior to the formation of a ternary polymerase complex by the assembly of PA in the infected cells. These results suggest that Hsp90 is involved in the assembly and nuclear transport of viral RNA polymerase subunits, possibly as a molecular chaperone for the polymerase subunits prior to the formation of a mature ternary polymerase complex.
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PMID:Involvement of Hsp90 in assembly and nuclear import of influenza virus RNA polymerase subunits. 1712 7

The transcription/replication activity of ribonucleoproteins derived from influenza A primary isolates of human (A/Paris/908/97) or avian origin (A/Mallard/Marquenterre/MZ237/83, A/Hong Kong/156/97) was compared upon reconstitution in mammalian or avian cells, using viral-like reporter RNAs synthesized under the control of the human and chicken RNA polymerase I promoters, respectively. In avian cells, transcription/replication activities were in the same range with all ribonucleoproteins tested. In human cells, ribonucleoproteins derived from A/Mallard/Marquenterre/MZ237/83 showed reduced transcription/replication activity and reduced NP binding to the PB1-PB2-PA complex (P) or to the isolated PB2 subunit, as compared to the ribonucleoproteins derived from A/Paris/908/97. Both defects were restored when PB2 residue Glu-627 was changed to a Lys. Ribonucleoproteins derived from the human A/Hong Kong/156/97 H5N1 isolate showed efficient NP-P interaction in human cells, and high levels of activity which were determined mostly by the PB2 and PA proteins. Our data suggest that PB2 might play a pivotal role in molecular interactions involving both the viral nucleoprotein and cellular proteins.
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PMID:Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells. 1727 Feb 30


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