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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)

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.
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PMID:Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly. 171 23

Yeast RNA polymerases A (I) and C (III) share a subunit called AC19. The gene encoding AC19 has been isolated from yeast genomic DNA using oligonucleotide probes deduced from peptide sequences of the isolated subunit. This gene (RPC19) contains an intron-free open reading frame of 143 amino acid residues. RPC19 is a single copy gene that maps on chromosome II and is essential for cell viability. The amino acid sequence contains a sequence motif common to the Escherichia coli RNA polymerase alpha subunit, the Saccharomyces cerevisiae AC40 and B44.5 subunits, the human hRPB33 product, and the CnjC conjugation-specific gene product of Tetrahymena. The 5'-upstream region contains a sequence element, the PAC box, that has been conserved in at least 10 genes encoding subunits of RNA polymerases A and C.
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PMID:RPC19, the gene for a subunit common to yeast RNA polymerases A (I) and C (III). 186 54

The cnjC gene from the protozoan Tetrahymena thermophila was completely sequenced. The deduced gene product was found to have significant sequence similarity to the yeast and prokaryotic RNA polymerase subunits involved with subunit assembly. Since cnjC is active only during the sexual stage (conjugation) of Tetrahymena's life cycle, these results indicate it may be part of a novel type of transcriptional control. The yeast proteins to which the Tetrahymena cnjC is homologous are the 40 kd protein of RNA polymerases I and III (coded for by gene RPC40) and the third-largest subunit of RNA polymerase II (coded for by gene RPB3). The degree of similarity of the cnjC protein to the two yeast subunits was found to be greater than the similarity of the two yeast subunits to each other. The alpha subunit of the core RNA polymerase from prokaryotes (coded for by gene rpoA) was found to have regions of similarity to the cnjC protein as well as to the subunits encoded by RPC40 and RPB3. Regions of high conservation among the four proteins are noted. The significance of these results is discussed.
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PMID:A conjugation-specific gene (cnjC) from Tetrahymena encodes a protein homologous to yeast RNA polymerase subunits (RPB3, RPC40) and similar to a portion of the prokaryotic RNA polymerase alpha subunit (rpoA). 211 40

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.
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PMID:RNA polymerase II subunit composition, stoichiometry, and phosphorylation. 218 13

To improve our understanding of RNA polymerase II, the gene that encodes its third-largest subunit, RPB3, was isolated from a lambda gt11 DNA library by using antibody probes. The RPB3 DNA sequence predicts a 318-amino-acid protein whose sequence was confirmed, in part, by microsequence analysis of the gel-purified RNA polymerase II subunit. RPB3 was found to be an essential single-copy gene that is tightly linked to HIS6 on chromosome IX. An RPB3 temperature-sensitive mutant that arrested growth after three to four generations at the restrictive temperature was isolated. When the mutant was shifted to the restrictive temperature, RNA polymerase II could no longer assemble, previously assembled functional enzyme was depleted, and mRNA levels were consequently reduced. These results demonstrate that RPB3 is an essential component of the mRNA transcription apparatus. Finally, the RPB3 protein is similar in sequence and length to RPC5, a subunit common to RNA polymerases I and III, suggesting that these subunits may play similar roles in RNA polymerases I, II, and III.
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PMID:RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus. 268 62

The diverse functions of Saccharomyces cerevisiae RNA polymerase II are partitioned among its 12 subunits, designated RPB1-RPB12. Although multiple functions have been assigned to the three largest subunits, RPB1, RPB2, and RPB3, the functions of the remaining smaller subunits are unknown. We have determined the function of one of the smaller subunits, RPB9, by demonstrating that it is necessary for accurate start site selection. Transcription in the absence of RPB9 initiates farther upstream at new and previously minor start sites both at the CYC1 promoter in vitro and at the CYC1, ADH1, HIS4, H2B-1, and RPB6 promoters in vivo. Immunoprecipitation of RNA polymerase II from cells lacking the RPB9 gene revealed that all of the remaining 11 subunits are assembled into the enzyme, suggesting that the start site defect is attributable solely to the absence of RPB9. In support of this hypothesis, we have shown that addition of wild-type recombinant RPB9 completely corrects for the start site defect seen in vitro. A mutated recombinant RPB9 protein, with an alteration in a metal-binding domain required for high temperature growth and accurate start site selection in vivo, was at least 10-fold less effective at correcting the start site defect in vitro. RPB9 appears to play a unique role in transcription initiation, as the defects revealed in its absence are distinct from those seen with mutants in RNA polymerase subunit RPB1 and factor e (TFIIB), two other yeast proteins also involved in start site selection.
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PMID:RNA polymerase II subunit RPB9 is required for accurate start site selection. 788 69

Mouse RNA polymerase I (or A) was purified from an ascites cell line MH134 to virtual homogeneity using a novel purification procedure and examined for subunit composition. In marked contrast to older purifications that reported 5-8 subunits, polymerase I was found to have 11 subunits with remarkable correspondence to those of yeasts. The cDNA encoding a 40-kDa subunit of this enzyme, designated RPA40, was isolated. It predicts a polypeptide of 355 amino acids (M(r) = 40,065) and is encoded by a single copy gene. Protein sequence analysis reveals that RPA40 is the homolog of yeast RPC40, having homology to alpha subunit of Escherichia coli RNA polymerase, yeast RPB3, and human RPB33 RNA polymerase II subunits. The high conservation of this subunit among distant eukaryotes and different RNA polymerases suggests functional importance of this protein as a core subunit.
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PMID:High conservation of subunit composition of RNA polymerase I(A) between yeast and mouse and the molecular cloning of mouse RNA polymerase I 40-kDa subunit RPA40. 792 37

The eukaryotic DNA-dependent RNA polymerase II (or B) is composed of 10 to 14 polypeptides ranging from 220 to 10 kDa. To gain further insight into the molecular structure and function of these subunits, we have undertaken the molecular cloning of nucleotide sequences corresponding to the human enzyme. The cDNAs of five subunits (hRPB220, hRPB140, hRPB33, hRPB25, and hRPB14.5) have been isolated. Using in situ hybridization, we show that the genes of these subunits have distinct chromosomal locations (17p13, 4q12, 16q13-q21, 19p13.3, and 19q12, respectively). Thus, if assembly of active polymerase molecules requires coordinated expression from these independent genes, mechanisms that ensure tight coregulation of the corresponding promoters must exist.
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PMID:Chromosomal localization of human RNA polymerase II subunit genes. 803 26

Two subunits in RNA polymerase II (e.g. RPB3 and RPB11 in yeast) and two subunits common to RNA polymerases I and III (e.g. AC40 and AC19 in yeast) contain one or two motifs related to the alpha subunit in prokaryotic RNA polymerases. We have sequenced two different cDNAs (AtRPB36a and AtRPB36b), the two corresponding genes from Arabidopsis thaliana that are homologs of yeast RPB3, and an Arabidopsis cDNA (AtRPB13.6) that is a homolog of yeast RPB11. The B36a subunit is the predominant B36 subunit associated with RNA polymerase II purified from Arabidopsis suspension culture cells, and this subunit has a stoichiometry of about 1. Results from protein association assays showed that the B36a and B36b subunits did not associate, but each of these subunits did associate with the B13.6 subunit in vivo and in vitro. Two motifs in the B36b subunit related to the prokaryotic alpha subunit were shown to be required for the in vitro interactions with the B13.6 subunit. Our results suggest that the B36 and B13.6 subunits associate to form heterodimers in Arabidopsis RNA polymerase II like the AC40 and AC19 heterodimers reported for yeast RNA polymerases I and III but unlike the B44 homodimers reported for yeast RNA polymerase II.
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PMID:Association between 36- and 13.6-kDa alpha-like subunits of Arabidopsis thaliana RNA polymerase II. 861 87

The cDNA encoding a protein that interacts with the mouse homologue of the yeast RNA polymerase II (polII) subunit, RPB11, and the human polII subunit, hRPB14, has been isolated by protein interaction cloning. Its deduced amino acid sequence has 96% homology to the human third largest polII subunit, hRPB33 [Pati and Weissman (1990) J. Biol. Chem. 265, 8400 8405]. Therefore, we conclude that the cloned cDNA encodes the mouse third largest polII subunit, mRPB31. Isolation of cDNA by protein interaction cloning provides evidence supporting the hypothesis, first proposed for human polII assembly [Pati (1994) Gene 145, 289-292], that the mRPB31/mRPB14 heterodimer, rather than the mRPB31 homodimer, forms in the mouse polII assembly. Indeed, in the yeast two-hybrid system, mRPB31 was shown to fail to form homodimer.
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PMID:Protein interaction cloning in yeast of the mouse third largest RNA polymerase II subunit, mRPB31. 903 5


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