Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 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.
Mol Cell Biol 1991 Sep
PMID:Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly. 171 23

An elongation block to RNA polymerase II transcription in exon 1 is a major regulatory step in expression of the murine adenosine deaminase (ADA) gene. Previous work in the laboratory identified abundant short transcripts with 3' termini in exon 1 in steady-state RNA from injected oocytes. Using a cell-free system to investigate the mechanism of premature 3' end formation, we found that polymerase II generates prominent ADA transcripts approximately 96 to 100 nucleotides in length which are similar to the major short transcripts found in steady-state RNA from oocytes injected with ADA templates. We have determined that these transcripts are the processed products of 108- to 112-nucleotide precursors. Precursor formation is (i) favored in reactions using circular templates, (ii) not the result of a posttranscriptional processing event, (iii) sensitive to low concentrations of Sarkosyl, and (iv) dependent on a factor(s) which is inactivated in crude extracts at 47 degrees C for 15 min. The cell-free system will allow further characterization of the template and factor requirements involved in the control of premature 3' end formation by RNA polymerase II.
Mol Cell Biol 1991 Nov
PMID:A heat-labile factor promotes premature 3' end formation in exon 1 of the murine adenosine deaminase gene in a cell-free transcription system. 171 27

RAP30/74 is a human general transcription factor that binds to RNA polymerase II and is required for initiation of transcription in vitro regardless of whether the promoter has a recognizable TATA box (Z. F. Burton, M. Killeen, M. Sopta, L. G. Ortolan, and J. F. Greenblatt, Mol. Cell. Biol. 8:1602-1613, 1988). Part of the amino acid sequence of RAP30, the small subunit of RAP30/74, has limited homology with part of Escherichia coli sigma 70 (M. Sopta, Z. F. Burton, and J. Greenblatt, Nature (London) 341:410-414, 1989). To determine which sigmalike activities of RAP30/74 could be attributed to RAP30, we purified human RAP30 and a RAP30-glutathione-S-transferase fusion protein that had been produced in E. coli. Bacterially produced RAP30 bound to RNA polymerase II in the absence of RAP74. Both partially purified natural RAP30/74 and recombinant RAP30 prevented RNA polymerase II from binding nonspecifically to DNA. In addition, nonspecific transcription by RNA polymerase II was greatly inhibited by RAP30-glutathione-S-transferase. DNA-bound RNA polymerase II could be removed from DNA by partially purified RAP30/74 but not by bacterially expressed RAP30. Thus, the ability of RAP30/74 to recruit RNA polymerase II to a promoter-bound preinitiation complex may be an indirect consequence of its ability to suppress nonspecific binding of RNA polymerase II to DNA.
Mol Cell Biol 1992 Jan
PMID:The general transcription factor RAP30 binds to RNA polymerase II and prevents it from binding nonspecifically to DNA. 172 6

To identify the DNA sequences required for initiation of transcription in archaea, the 5'-flanking region of the tRNA(Val) gene of Methanococcus vannielii was modified by deletions, restructuring and site-directed mutagenesis, and the tRNA encoding sequence was replaced by a fortuitous Escherichia coli sequence. The effects of these mutations on promoter function were tested in an homologous cell-free transcription system. The DNA region from position -35 to +9 relative to the transcription start site was sufficient for maximal initiation of cell-free transcription. Removal of the DNA region between -35 and -30 reduced initiation by a factor of 2. Deletions extending to position -24 almost completely abolished specific transcription. Analysis of 16 site-specific mutations in the region from -33 to +2 provided evidence that a conserved A + T-rich sequence (TATA box), centered at -25, is essential for initiation of transcription. Single point mutations in six positions of the TATA box reduced initiation of transcription from 0.2 to 0.01 of wild-type levels. A second conserved motif at the transcription start site (consensus ATGC) could be replaced by some sequences containing a pyrimidine-purine dinucleotide but appeared necessary for a maximal rate of gene transcription. Mutations altering the spacing between the two conserved elements demonstrated that initiation occurs at a strictly defined distance of 22 to 27 base-pairs downstream from the TATA box. Our results support the conclusion that the TATA box is the major DNA region mediating promoter recognition, influencing the efficiency of transcription and specifying the site of transcription initiation. This Methanococcus promoter element closely resembles in structure and function the TATA box of promoters of eukaryotic protein-encoding genes transcribed by RNA polymerase II.
J Mol Biol 1991 Dec 05
PMID:Control regions of an archaeal gene. A TATA box and an initiator element promote cell-free transcription of the tRNA(Val) gene of Methanococcus vannielii. 174 92

Transcription of the rabbit alpha-globin gene begins primarily at the cap site, although some upstream start sites are also observed. Analysis by RNA polymerase run-on assays in nuclei shows that transcription continues at a high level past the polyadenylation site, after which the polymerase density actually increases in a region of about 400 nucleotides, followed by a gradual decline over the 700 nucleotides. These features are also observed in the transcription unit of the rabbit beta-globin gene. The region with the unexpectedly high nascent RNA hybridization signal in the 3' flank contains a conserved sequence, KGCAGCWGGR (K = G or T, W = A or T, R = A or G), followed by an inverted repeat. The inverted repeat (perhaps with the conserved sequence) may be a pause site for RNA polymerase II, thus accounting for the increase in polymerase density. This sequence and inverted repeat are found in the 3' flank of several globin genes and the simian virus 40 (SV40) early genes, as well as in the regions implicated in pausing or termination of transcription of eight different genes. Deletion of the conserved sequence and inverted repeat from the 3' flank of the SV40 early region causes a small increase in the levels of transcription downstream from this site. Replacement with the conserved sequence and inverted repeat from the rabbit alpha-globin gene causes an accumulation of polymerases, supporting the hypothesis that polymerases pause at this site. This proposed pause site may affect the efficiency of termination at some sites further downstream, perhaps by loss of a processivity factor.
J Mol Biol 1991 Jul 20
PMID:An apparent pause site in the transcription unit of the rabbit alpha-globin gene. 185 60

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.
Mol Cell Biol 1991 Sep
PMID:Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae. 187 38

A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.
Mol Cell Biol 1991 May
PMID:Sequence requirements for premature transcription arrest within the first intron of the mouse c-fos gene. 190 50

To analyze the transcriptional control regions of Drosophila melanogaster household genes, we have characterized the promoter of the gene coding for the second-largest subunit of RNA polymerase II (DmRP140). Analysis of cDNA revealed that the coding region of the protein extends beyond the originally assumed transcription start point (tsp) and deduced translation start codon [Falkenburg et al., J. Mol. Biol. 195 (1987) 929-937] and that the tsp determined previously corresponds to an intron/exon boundary of an additional intron. Upstream of the polII gene we found a transcription unit that is transcribed in the opposite direction. The initiating ATGs of the two genes are only 467 nucleotides (nt) apart. The untranslated region is extremely A + T-rich (88%) but none of the transcription units is preceded by a canonical TATA element. It does not feature any other known nt sequence motifs thought to be necessary for the basic transcriptional machinery; yet, this region functions as a bidirectional promoter: a central 309-bp fragment directs transcription of a reporter gene in transiently transfected Drosophila culture cells in both orientations. The gene coding for the second-largest subunit of RNA polymerase II of Drosophila virilis (DvRP140) was isolated and partially analyzed. The gene is located on the second chromosome at 22F/23A which corresponds to the position determined for D. melanogaster.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Analysis of the promoter region of the housekeeping gene DmRP140 by sequence comparison of Drosophila melanogaster and Drosophila virilis. 193 28

We have isolated cDNA and genomic clones of Drosophila melanogaster by cross-hybridization with a 658 bp fragment of the yeast gene coding for the second-largest subunit of RNA polymerase III (RET1). Determination of the sequence by comparison of genomic and cDNA regions reveals an ORF of 3405 nucleotides which is interrupted in the genomic sequence by an intron of 48 bp. The deduced polypeptide consists of 1135 amino acids with a calculated molecular weight of 128 kDa. The protein sequence shows the same conserved regions of homology as those observed for all the second-largest subunits of RNA polymerases cloned so far. The gene (DmRP128) obviously codes for a second-largest subunit of an RNA polymerase which is different from DmRP140 and DmRP135. We have purified three distinct RNA polymerase activities from D. melanogaster. By using specific RNA polymerase inhibitors in enzyme assays and by comparing their subunit composition we were able to distinguish between RNA polymerase I, II, and III. RNA polymerase preparations of D. melanogaster were blotted and the second-largest subunits were identified with antibodies raised against polypeptides expressed from DmRP128 and DmRP135. Anti-DmRP135 antibodies react strongly with the second-largest subunit of RNA polymerase I but do not react with the respective subunits of RNA polymerase II and III. The second-largest subunit of RNA polymerase III is only recognized by anti-DmRP128. Previously, we have claimed that DmRP135 codes for the second-largest subunit of RNA polymerase III.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Gen Genet 1991 Sep
PMID:Identification of the genes coding for the second-largest subunits of RNA polymerases I and III of Drosophila melanogaster. 191 Jan 49

The mammalian ribosomal RNA gene promoters exhibit a conserved sequence between positions +1 and +16 that shows a high degree of homology to the response element for glucocorticoids and progestins (GRE/PRE). These sequences bind specifically the glucocorticoid receptor and the progesterone receptor (PR) albeit with lower affinity than a canonical GRE/PRE. Because steroid hormones are known to affect expression of the ribosomal genes, we tested the influence of hormone receptors on the activity of the ribosomal RNA gene promoter in a cell-free transcription assay. Preparations of PR that induce transcription from the mouse mammary tumour virus (MMTV) promoter do not stimulate but slightly inhibit transcription from the ribosomal RNA gene promoter. This weak negative effect is not mediated through binding to the hypothetical GRE/PRE as a mutant promoter that does not bind receptor is equally repressed. Introduction of the functional MMTV GRE/PRE upstream of the basal ribosomal RNA gene promoter does not enhance its transcription in the presence of an active PR. Thus, RNA polymerase I transcription cannot be stimulated in vitro by cis elements and regulatory proteins that are active in RNA polymerase II transcription.
J Steroid Biochem Mol Biol 1991 Oct
PMID:Neither the endogenous nor a functional steroid hormone receptor binding site transactivate the ribosomal RNA gene promoter in vitro. 191 32


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