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)

RNA polymerase II is a multisubunit enzyme composed of two large subunits of molecular weight in excess of 100,000 and a collection of 8-10 smaller subunits. The largest subunit, designated IIa, contains at its carboxyl terminus a highly repetitive domain consisting of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Extensive phosphorylation within this COOH-terminal domain (CTD) gives rise to subunit IIo which has a markedly reduced mobility in SDS-polyacrylamide gel electrophoresis (PAGE) relative to subunit IIa. Recent evidence suggests that RNA polymerase IIA, containing an unphosphorylated CTD, is involved in preinitiation complex assembly, whereas RNA polymerase IIO is involved in elongation. Consequently, CTD phosphorylation is thought to occur after RNA polymerase II has bound to the promoter by a protein kinase that stably associates with the preinitiation complex. We present here the partial purification and characterization of two distinct CTD kinases from a HeLa cell transcription extract. These CTD kinases, designated CTDK1 and CTDK2, are fractionated by chromatography on Mono Q. CTDK1 catalyzes the incorporation of approximately 33 pmol of phosphate/pmol of calf thymus RNA polymerase subunit IIa, almost exclusively on serine. CTDK2 catalyzes the incorporation of approximately 50 pmol of phosphate/pmol of calf thymus subunit IIa, predominantly on serine; appreciable phosphate transfer onto threonine is also observed. Phosphorylation by CTDK2, but not CTDK1, results in a complete mobility shift in SDS-PAGE of subunit IIa to the position of IIo. CTDK1 can utilize ATP, dATP, or GTP as phosphate donor, whereas CTDK2 can utilize only ATP or dATP. The apparent Km for ATP is 30 microM for CTDK1 and 60 microM for CTDK2. CTDK1 and CTDK2 also differ in their protein substrate specificity. CTDK1 phosphorylates casein whereas CTDK2 does not. Neither kinase phosphorylates phosvitin or histone H1 to an appreciable extent. CTDK1 and CTDK2 do not appear to be related to cdc2 kinases as determined by their inability to phosphorylate H1 and their failure to react with antibodies directed against the cdc2 kinase. These results establish that a partially fractionated HeLa transcription extract contains two distinct CTD kinases that differ in their nucleotide requirements and in their patterns of CTD phosphorylation.
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PMID:Partial purification and characterization of two distinct protein kinases that differentially phosphorylate the carboxyl-terminal domain of RNA polymerase subunit IIa. 841 77

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication.
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PMID:African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases. 850 38

The characterization of RNA polymerase subunit genes has revealed that some subunits are shared by the three nuclear enzymes, some are homologous, and some are unique to RNA polymerases I, II, or III. We report here the isolation and characterization of the yeast RNA polymerase II subunit RPB11, which is encoded by a single copy RPB11 gene located directly upstream of the topoisomerase I gene, TOPI, on chromosome XV. The sequence of the gene predicts an RPB11 subunit of 120 amino acids (13,600 daltons), only two amino acids shorter than the RPB9 polypeptide, that co-migrates with RPB11 under most SDS-PAGE conditions, RPB11 was found to be an essential gene that encodes a protein closely related to an essential subunit shared by RNA polymerases I and III, AC19. RPB11 contains a 19 amino acid segment found in three other yeast RNA polymerase subunits and the bacterial RNA polymerase subunit alpha. Some mutations that affect RNA polymerase assembly map within this segment, suggesting that this region may play a role in subunit interactions. As the isolation of RPB11 completes the isolation of known yeast RNA polymerase II subunit genes, we briefly summarize the salient features of these twelve genes and the polypeptides that they encode.
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PMID:Yeast RNA polymerase II subunit RPB11 is related to a subunit shared by RNA polymerase I and III. 850 29

Subunit alpha of prokaryotic RNA polymerases plays key roles in protein-protein contacts for both subunit assembly and transcription activation. To gain an insight into the roles of subunit 3, the eukaryotic homologue of alpha, temperature-sensitive mutants of the fission yeast, Schizosaccharomyces pombe, have been isolated after transformation of the mutagenized rpb3 gene encoding mutant subunit 3 of RNA polymerase II. A total of 68 ts mutants were classified into two groups: mutants comprising one group ceased growing immediately after a temperature up-shift, while mutants comprising the other group exhibited delayed growth arrest at high temperatures. RNA polymerase II partially purified from Ts54, one of the group 2 mutants, was thermolabile in vitro, as measured by a non-specific transcription assay. This mutant carries double mutations in domain A of subunit 3, and thus can be used as a reference mutant of RNA polymerase II.
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PMID:Isolation of thermolabile mutant RNA polymerase II from fission yeast Schizosaccharomyces pombe with mutations in the subunit 3 gene. 853 15

Two myxoma virus transient dominant selection vectors were constructed and used to generate recombinant viruses expressing single and double foreign gene insertions from intergenic sites. The intergenic insertion sites were located between the myxoma virus genes MJ2 (thymidine kinase) and MJ2a, and MA24 (beta-subunit RNA polymerase) and MA27 (fusion protein) located approximately 60 and 113 kb from the left-end of the viral genome, respectively. Recombinant myxoma viruses expressing the lacZ gene from either intergenic insertion site retained wild-type virulence. However, expression of the gus gene reduced the virulence of the recombinant viruses in vivo. Northern blot analysis indicated that the major late mRNAs encoding the viral RNA polymerase subunit and fusion protein are both of discrete size. Insertion of a foreign gene under the control of a synthetic late promoter between the MA24 and MA27 genes results in a specific-sized major late transcript for the inserted foreign gene. The MA27 gene transcripts directed by these recombinant viruses are heterogeneous in size, implying the typical pattern of poxvirus late transcription by random 3'-termination prior to polyadenylation. The transcription studies suggest signals located downstream of the insertion site direct 3'-processing of late transcripts irrespective of the gene immediately upstream.
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PMID:Construction of recombinant myxoma viruses expressing foreign genes from different intergenic sites without associated attenuation. 875 1

We have cloned the gene encoding RNA polymerase alpha subunit from a gene library of deep-sea barophilic bacterium strain DB6705. The clone contains the genes for ribosomal protein S4, RNA polymerase subunit alpha and ribosomal protein L17 in this order. The alpha gene has 328 amino acids and a molecular mass of 36 100 Da with 86.9% identity to Escherichia coli alpha gene. Differences between the two sequences were mainly in the N-terminal portion of the alpha subunit, which is involved in the assembly of the core RNA polymerase; while the 87 C-terminal residues, which form a region involved in contact with some positive regulators and rrnB P1 promoter region called UP-element, were identical in the both strain. Plasmid encoding the alpha subunit with an N-terminal hexahistidine tag was constructed. Using the plasmid, the recombinant fusion alpha subunit was overexpressed and successfully purified to near homogeneity.
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PMID:Molecular cloning of the gene encoding RNA polymerase alpha subunit from deep-sea barophilic bacterium. 876 26

The gene (POLR2L) encoding a 7.6-kDa subunit (hRPB7.6) of human RNA polymerase has been cloned. It compromises two exons, 116 and 227 bp, respectively, interspaced with an intron of about 2.1 kb. This gene, whose localization has been assigned to the short arm of chromosome 11 (position 11p15), is transcribed in HeLa cells as one major messenger RNA, which encodes a 67-residue polypeptide (7645 Da) that shares strong homologies with the corresponding subunits of other eukaryotic and archaeal RNA polymerase subunits. Like its yeast counterpart (ABC10 beta, encoded by the RPB10 gene), the hRPB7.6 subunit may be shared by all three classes of human nuclear RNA polymerase. Cysteine residues characteristic of an atypical zinc-binding domain are conserved in the homologous sequences of all six species analyzed. A small, related RNA polymerase subunit from vaccinia virus exhibits an identical set of cysteines, suggesting that these residues may be contribute to a crucial function in the multimeric RNA polymerases.
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PMID:The gene (POLR2L) encoding the hRPB7.6 subunit of human RNA polymerase. 878 24

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.
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PMID:The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function. 879 Mar 57

We have previously isolated a mouse RPA40 (mRPA40) cDNA encoding the 40-kDa subunit of mouse RNA polymerase I and demonstrated that mRPA40 is a mouse homolog of the yeast subunit AC40, which is a subunit of RNA polymerases I and III, having a limited homology to bacterial RNA polymerase subunit alpha (Song, C. Z., Hanada, K., Yano, K., Maeda, Y., Yamamoto, K., and Muramatsu, M. (1994) J. Biol. Chem. 269, 26976-26981). In an extension of the study we have now cloned mouse RPA16 (mRPA16) cDNA encoding the 16-kDa subunit of mouse RNA polymerase I by a yeast two-hybrid system using mRPA40 as a bait. The deduced amino acid sequence shows 45% identity to the yeast subunit AC19 of RNA polymerases I and III, known to associate with AC40, and a local similarity to bacterial alpha subunit. We have shown that mRPA40 mutants failed to interact with mRPA16 and that neither mRPA16 nor mRPA40 can interact by itself in the yeast two-hybrid system. These results suggest that higher eukaryotic RNA polymerase I conserves two distinct alpha-related subunits that function to associate with each other in an early stage of RNA polymerase I assembly.
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PMID:Mouse RNA polymerase I 16-kDa subunit able to associate with 40-kDa subunit is a homolog of yeast AC19 subunit of RNA polymerases I and III. 895 28

In transcription initiation, the DNA strands must be separated to expose the template to RNA polymerase. As the closed initiation complex is converted to an open one, specific protein-DNA interactions involving bases of the nontemplate strand form and stabilize the promoter complex in the region of unwinding. Specific interaction between RNA polymerase and the promoter in Escherichia coli was detected and quantified as the binding affinity of nontemplate oligonucleotide sequences. The RNA polymerase subunit sigma factor 70 contacted the bases of the nontemplate DNA strand through its conserved region 2; a mutation that affected promoter function altered the binding affinity of the oligonucleotide to the enzyme.
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PMID:Promoter recognition as measured by binding of polymerase to nontemplate strand oligonucleotide. 915 85


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