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)

The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein, where L is believed to possess all the enzymatic activities necessary for viral transcription and replication. The alignment of amino acid sequences of L proteins from negative-sense RNA viruses shows six regions, designated domains I-VI, of good conservation which have been proposed to be important for the various enzymatic activities of the polymerase. To directly address the role(s) of domains II and III, site-directed mutations were constructed by the substitution of multiple amino acids at 13 highly or mostly conserved residues. Analysis of in vitro viral transcription and replication showed that the majority of the mutations completely inactivated the L protein for all aspects of RNA synthesis, thus conservation correlated with the essential nature of the amino acid. At some positions different phenotypes, from inactivation to partial activities, were observed which depended on the nature of the amino acid that was substituted. Two mutants, K543R and K666V, could synthesize some leader RNA, but were defective in mRNA synthesis and replication. K666R and G737E had significantly reduced replication compared to transcription in vitro, but replicated genome RNA much more efficiently in vivo. K666A gave transcription, but no replication. Representative inactive L mutants, however, were still able to bind P protein and the polymerase complex was capable of binding nucleocapsids, so the defect appeared to be in the initiation of RNA synthesis.
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PMID:Different substitutions at conserved amino acids in domains II and III in the Sendai L RNA polymerase protein inactivate viral RNA synthesis. 1249 Apr 11

The PITSLRE protein kinases, hereafter referred to as CDK11 because of their association with the cyclin L regulatory partner, belong to large molecular weight protein complexes that contain RNA polymerase II. These CDK11(p110) complexes have been reported to influence transcription as well as interact with the general pre-mRNA-splicing factor RNPS1. Some of these complexes may also play a role in pre-mRNA splicing. Using a two-hybrid interactive screen, the splicing protein 9G8 was identified as an in vivo partner for CDK11(p110). The identification of several splicing-related factors as CDK11(p110) interactors along with the close relationship between transcription and splicing indicated that CDK11(p110) might influence splicing activity directly. Immunodepletion of CDK11(p110) from splicing extracts greatly reduced the appearance of spliced products using an in vitro assay system. Moreover, the re-addition of these CDK11(p110) immune complexes to the CDK11(p110)-immunodepleted splicing reactions completely restored splicing activity. Similarly, the addition of purified CDK11(p110) amino-terminal domain protein was sufficient to inhibit the splicing reaction. Finally, 9G8 is a phosphoprotein in vivo and is a substrate for CDK11(p110) phosphorylation in vitro. These data are among the first demonstrations showing that a CDK activity is functionally coupled to the regulation of pre-mRNA-splicing events and further support the hypothesis that CDK11(p110) is in a signaling pathway that may help to coordinate transcription and RNA-processing events.
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PMID:CDK11 complexes promote pre-mRNA splicing. 1250 Dec 47

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the fusion (F)- and hemagglutinin-neuraminidase genes in a full-length cDNA clone of NDV. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in embryonated eggs and the allantoic fluid was used to infect cells. Northern blot analysis of RNA isolated from infected cells demonstrated the proper transcription of the introduced GFP-mRNA. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDVGFP1) expressing the reporter gene. The expression of the heterologous gene was maintained stably for at least five passages in embryonated eggs. The replication kinetics in embryonated eggs and pathogenicity in chickens of rNDVGFP1 did not differ significantly from that of the parent virus. Using GFP autofluorescence, virus infected cells could be tracked easily in native preparations, organ explants and primary tracheal cell cultures. Taken together, these data demonstrate the use of GFP-expressing recombinant NDV for analysis of NDV dissemination and pathogenesis and indicate the potential usefulness of NDV as a vaccine vector.
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PMID:Characterization of a recombinant Newcastle disease virus expressing the green fluorescent protein. 1256 50

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is heavily phosphorylated during the transition from transcription initiation to the establishment of an elongation-competent transcription complex. FCP1 is the only phosphatase known to be specific for the CTD of the largest subunit of RNAPII, and its activity is believed to be required to reactivate RNAPII, so that RNAPII can enter another round of transcription. We demonstrate that FCP1 is a phosphoprotein, and that phosphorylation regulates FCP1 activities. FCP1 is phosphorylated at multiple sites in vivo. The CTD phosphatase activity of phosphorylated FCP1 is stimulated by TFIIF, whereas dephosphorylated FCP1 is not. In addition to its role in the recycling of RNAPII, FCP1 also affects transcription elongation. Phosphorylated FCP1 is more active in stimulating transcription elongation than the dephosphorylated form of FCP1. We found that only phosphorylated FCP1 can physically interact with TFIIF. We set out to purify an FCP1 kinase from HeLa cells and identified casein kinase 2, which, surprisingly, displayed a negative effect on FCP1-associated activities.
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PMID:The C-terminal domain phosphatase and transcription elongation activities of FCP1 are regulated by phosphorylation. 1259 39

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are grouped in the Novirhabdovirus genus within the family Rhabdoviridae. There are many similarities between these two viruses including the lengths of the leader and trailer regions and the homologies of the terminal sequences. We have developed two systems in which IHNV and VHSV minigenomes encoding the marker green fluorescent protein (GFP) can be expressed from plasmids by T7 RNA polymerase. These negative sense minigenome RNAs can be replicated, transcribed and packaged into infectious particles when coinfected with homologous helper viruses. After infection of the minigenome transfected BHK-T7 cells by heterologous helper viruses GFP expression was observed, but packaging of the minigenome RNAs into virus particles did not occur. Packaging of chimeric minigenomes by IHNV and VHSV was also not observed. Cotransfections of the negative sense minigenome plasmids with plasmids encoding nucleoprotein (N), phosphoprotein (P) and RNA polymerase (L) of IHNV and VHSV were carried out in all combinations. Minigenome constructs were expressed only after cotransfection with a set of helper plasmids (N, P and L) all originated from one virus. These results indicate that the cis-acting elements responsible for the encapsidation and transcription were recognized by the homologous and heterologous helper proteins, but packaging of the minigenome RNAs required homologous helper viruses.
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PMID:Recognition of cis-acting elements of infectious haematopoietic necrosis virus and viral hemorrhagic septicemia virus by homologous and heterologous helper proteins. 1272 45

Borna disease virus (BDV) is an enveloped virus. Its non-segmented, negative-stranded RNA genome has the coding capability for six main polypeptides and has an organization characteristic of members of the order Mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae. Here, the establishment of a reverse genetics system for BDV is described. Intracellular synthesis of a BDV RNA analogue or minigenome (MG) from a plasmid was driven by RNA polymerase I. Co-transfection with plasmids expressing the BDV polymerase (L), nucleoprotein (N) and phosphoprotein (P) under the control of RNA polymerase II allowed for BDV MG replication and expression. This process depended on a delicate N:P ratio, whereas the L:P ratio was less critical. Two isoforms of N, Np40 and Np38, are present in BDV-infected cells but only Np40 was strictly required for virus polymerase activity. BDV p10 polypeptide encoded by the P gene exhibited a strong inhibitory effect on BDV MG expression.
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PMID:A reverse genetics system for Borna disease virus. 1457 15

The RNA polymerase sigma factor sigma(F) is a developmental regulatory protein that is activated in a cell-specific manner following the formation of the polar septum during the process of spore formation in the bacterium Bacillus subtilis. Activation of sigma(F) depends on the membrane-bound phosphatase SpoIIE, which localizes to the septum, and on the formation of the polar septum itself. SpoIIE is responsible for dephosphorylating and thereby activating the phosphoprotein SpoIIAA, which, in turn, triggers the release of sigma(F) from the anti-sigma(F) factor SpoIIAB. Paradoxically, however, the presence of unphosphorylated SpoIIAA is insufficient to cause sigma(F) activation as SpoIIAA reaches substantial levels in mutants blocked in polar septation. We now describe mutants of SpoIIE, SpoIIAA, and SpoIIAB that break the dependence of sigma(F) activation on polar division. Analysis of these mutants indicates that unphosphorylated SpoIIAA must reach a threshold concentration in order to trigger the release of sigma(F) from SpoIIAB. Evidence is presented that this threshold is created by the action of SpoIIAB, which can form an alternative, long lived complex with SpoIIAA. We propose that formation of the SpoIIAA-SpoIIAB complex serves as a sink that traps SpoIIAA in an inactive state and that only when unphosphorylated SpoIIAA is in excess to the sink does activation of sigma(F) take place.
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PMID:A threshold mechanism governing activation of the developmental regulatory protein sigma F in Bacillus subtilis. 1474 53

The RNA dependent RNA polymerase of Rinderpest virus consists of two subunits-the large protein (L) and the phosphoprotein (P), where L is thought to be responsible for the catalytic activities in association with P protein which plays multiple roles in transcription and replication. The nucleocapsid protein (N) is necessary for encapsidation of genomic RNA, which is required as N-P complex. To understand the different steps of transcription and replication as well as the roles played by the three proteins, an in vitro reconstitution system for RNA synthesis is necessary which is not available for any morbillivirus. We describe here, an in vitro reconstitution system for transcription and replication of Rinderpest virus utilizing a synthetic, positive sense N-RNA minigenome template, free of endogenous viral polymerase proteins and recombinant viral proteins (P+L and P+N) expressed in insect cells by recombinant baculoviruses. We show that although L-P complex is sufficient to synthesize negative sense minigenome RNA, soluble N protein is necessary for encapsidation of RNA as well as synthesis of (+) sense leader RNA and (+) sense minigenome RNA.
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PMID:Development of a reconstitution system for Rinderpest virus RNA synthesis in vitro. 1474 78

Despite the crucial importance of mitochondrial transcription, knowledge of its regulation is poor. Therefore, characterization of mammalian mitochondrial transcription termination factor (mTERF) functionality and regulation is of fundamental biological interest in order to understand the regulation of mitochondrial transcription. Here we report that mTERF is the first protein having a role in mammalian mitochondrial gene expression that appears to be controlled by phosphorylation. Recombinant mature rat mTERF protein has specific DNA-binding capacity for the sequence required for transcription termination. Furthermore, unlike recombinant human mTERF, the rat protein bound to its mitochondrial DNA binding site promotes the termination of transcription initiated with heterologous RNA polymerase. Interestingly, mTERF is a phosphoprotein with four phosphate groups, and while the DNA-binding activity of mTERF is unaffected by the phosphorylation/dephosphorylation state, only the phosphorylated form of the protein is active for termination activity. Moreover, natural human mTERF is also a phosphoprotein and its termination activity is inhibited by dephosphorylation. These data suggest that mTERF functioning in vivo is regulated by phosphorylation.
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PMID:Phosphorylation of rat mitochondrial transcription termination factor (mTERF) is required for transcription termination but not for binding to DNA. 1508 85

The RNA-dependent RNA polymerase complex of respiratory syncytial virus (RSV) is composed of the large polymerase (L), the phosphoprotein (P), the nucleocapsid protein (N) and the co-factors M2-1 and M2-2. The P protein plays a central role within the replicase-transcriptase machinery, forming homo-oligomers and complexes with N and L. In order to study P-P and N-P complexes, and the role of P phosphorylation in these interactions, the human RSV P and N proteins were expressed in E. coli as His-tagged or GST-fusion proteins. The non-phosphorylated status of recombinant P protein was established by mass spectrometry. GST-P and GST-N fusion proteins were able to interact with RSV proteins extracted from infected cells in a GST pull-down assay. When co-expressed in bacteria, GST-P and His-P were co-purified by glutathione-Sepharose affinity, showing that the RSV P protein can form oligomers within bacteria. This result was confirmed by chemical cross-linking experiments and gel filtration studies. The P oligomerization domain was investigated by a GST pull-down assay using a series of P deletion constructs. This domain was mapped to a small region situated in the central part of P (aa 120-150), which localized in a computer-predicted coiled-coil domain. When co-expressed in bacteria, RSV N and P proteins formed a soluble complex that prevented non-specific binding of N to bacterial RNA. Therefore, RSV P protein phosphorylation is not required for the formation of P-P and N-P complexes, and P controls the RNA binding activity of N.
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PMID:Biochemical characterization of the respiratory syncytial virus P-P and P-N protein complexes and localization of the P protein oligomerization domain. 1516 49


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