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Query: UNIPROT:P06889 (Mol)
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Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.
Mol Cell Biol 1992 Sep
PMID:A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo. 150 99

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.
Mol Cell Biol 1992 Sep
PMID:Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II. 150 10

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3'-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to alpha-amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.
Plant Mol Biol 1992 Sep
PMID:Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNA polymerase III. 151 Nov 42

The cDNA of the second largest subunit of RNA polymerase II (or B) from HeLa cells has been cloned and sequenced. A predicted amino acid sequence of 1174 residues (calculated molecular mass of 133,896 Da) was derived from the longest open reading frame and compared to the sequences of homologous subunits of polymerases of eukaryotic, archaeal and bacterial origin. After optimal alignment, about 16% of the residues were found to be conserved throughout evolution, from human to Escherichia coli. About 2/3 of the overall length of the conserved domains delineated by these residues are clustered within the C-terminal half of the human polypeptide, whereas the remaining is spread over its N-terminal half. The putative functional significance of these conserved domains is discussed.
J Mol Biol 1992 Aug 20
PMID:Primary structure of the second largest subunit of human RNA polymerase II (or B). 151 60

The promoters of both RNA polymerase II- and RNA polymerase III-transcribed small nuclear RNA (snRNA) genes contain an essential and highly conserved proximal sequence element (PSE) approximately 55 bp upstream from the transcription start site. In addition, the upstream enhancers of all snRNA genes contain binding sites for octamer-binding transcription factors (Octs), and functional studies have indicated that the PSE and octamer elements work cooperatively. The present study has identified and characterized a novel transcription factor (designated PTF) which specifically binds to the PSE sequence of both RNA polymerase II- and RNA polymerase III-transcribed snRNA genes. PTF binding is markedly potentiated by Oct binding to an adjacent octamer site. This potentiation is effected by Oct-1, Oct-2, or the conserved POU domain of these factors. In agreement with these results and despite the independent binding of Octs to the promoter, PTF and Oct-1 enhance transcription from the 7SK promoter in an interdependent manner. Moreover, the POU domain of Oct-1 is sufficient for significant in vitro activity in the presence of PTF. These results suggest that essential activation domains reside in PTF and that the potentiation of PTF binding by Octs plays a key role in the function of octamer-containing snRNA gene enhancers.
Mol Cell Biol 1992 Jul
PMID:Oct-1 and Oct-2 potentiate functional interactions of a transcription factor with the proximal sequence element of small nuclear RNA genes. 153 87

The Xenopus tropicalis U6 gene is very poorly transcribed both when introduced into human cells by transfection, and in human cell-free extracts. By analysis of hybrid promoters constructed from human and Xenopus sequences in various combinations, we show that species specificity is mediated by the proximal sequence elements (PSEs) of the promoters. We demonstrate the PSE-dependence of U6 transcription in a fractionated extract of HeLa cells. One of the fractions required for transcription contains an activity designated PSE-binding protein (PBP), previously shown to bind to the PSE of the mouse U6 gene. Binding of PBP to various wild-type and hybrid U6 PSE sequences correlates with their activity in transcription in HeLa cell extracts. This provides strong evidence that PBP is the PSE-binding factor involved in U6 transcription. In addition, it suggests that the differential affinities of the promoters for PBP is responsible for the observed species specificity. The divergence between U snRNA promoters in different species contrasts with the relatively strong conservation of other families of RNA polymerase II and III transcribed gene promoters. Possible mechanisms by which this diversity could be generated are discussed.
J Mol Biol 1992 Feb 20
PMID:Proximal sequence element factor binding and species specificity in vertebrate U6 snRNA promoters. 153 2

A small, divergently transcribed gene is located 500 bp upstream of the suppressor of Hairy-wing locus of Drosophila melanogaster. Sequencing of a full-length cDNA clone of the predominant 850-nucleotide transcript reveals that this gene encodes a 15,100-Da protein with high homology to a subunit of RNA polymerase II. The RpII15 protein is 46% identical to the RPB9 protein of Saccharomyces cerevisiae, one of the smallest subunits of RNA polymerase II from that species. Among those identical residues are four pairs of cysteines whose spacing is suggestive of two metal-binding "finger" domains. The gene is expressed at all developmental stages and in all tissues. Two deletions within the RpII15 gene are multiphasic lethal deletions, with accumulation of dead animals commencing at the second larval instar. Ovary transplantation experiments indicate that survival of mutant animals to this stage is due to the persistence of maternal gene product throughout embryogenesis and early larval development. The RpII15 gene product is thus necessary for viability of D. melanogaster.
Mol Cell Biol 1992 Mar
PMID:The RNA polymerase II 15-kilodalton subunit is essential for viability in Drosophila melanogaster. 154 24

Both genes encoding U3 small nuclear RNA (snRNA) from the budding yeast Saccharomyces cerevisiae were recently shown to be interrupted by introns of the type removed by the pre-mRNA splicing machinery. We previously described one of the two U3 genes from the fission yeast Schizosaccharomyces pombe. In the present work, the second S. pombe U3 coding sequence was identified, and direct RNA sequence analysis was used to show that neither the U3A nor the U3B gene from this organism contains an intervening sequence. Our data also demonstrate that, as expected, the two RNAs exhibit great primary- and secondary-structure conservation. These similarities are not likely to be the result of a recent gene duplication or conversion event, because the DNA sequences flanking the U3A and U3B genes have diverged substantially. A notable exception is a 19-bp block, centered 36 nucleotides upstream from the transcriptional start site, in which the two loci match in 15 positions; this motif may represent an RNA polymerase II upstream regulatory element, because related sequences are found preceding fission yeast U1, U2, U4, and U5 snRNA genes. The significance of a short conserved sequence just downstream of the U3A and U3B genes is unknown, as it is not found 3' to other snRNA coding sequences in S. pombe. The 5' one-third of U3B RNA can be folded into a dual hairpin structure, as we previously proposed for Schizosaccharomyces pombe U3A and for other lower eukaryotic U3 homologues. Quantitation of fission yeast U3A and U3B indicates that, in contrast to snR17A and B in Saccharomyces cerevisiae, these RNAs accumulate to similar levels.
Mol Biol Evol 1992 Mar
PMID:The two similarly expressed genes encoding U3 snRNA in Schizosaccharomyces pombe lack introns. 156 Jul 65

We have examined elongation by RNA polymerase II initiated at a promoter and have identified two classes of elongation complexes. Following initiation at a promoter, all polymerase molecules enter an abortive mode of elongation. Abortive elongation is characterized by the rapid generation of short transcripts due to pausing of the polymerase followed by termination of transcription. Termination of the early elongation complexes can be suppressed by the addition of 250 mM KCl or 1 mg of heparin per ml soon after initiation. Elongation complexes of the second class carry out productive elongation in which long transcripts can be synthesized. Productive elongation complexes are derived from early paused elongation complexes by the action of a factor which we call P-TEF (positive transcription elongation factor). P-TEF is inhibited by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole at concentrations which have no effect on the initiation of transcription. By using templates immobilized on paramagnetic particles, we show that isolated preinitiation complexes lack P-TEF and give rise to transcription complexes which can carry out only abortive elongation. The ability to carry out productive elongation can be restored to isolated transcription complexes by the addition of P-TEF after initiation. A model is presented which describes the role of elongation factors in the formation and maintenance of elongation complexes. The model is consistent with the available in vivo data concerning control of elongation and is used to predict the outcome of other potential in vitro and in vivo experiments.
Mol Cell Biol 1992 May
PMID:Control of formation of two distinct classes of RNA polymerase II elongation complexes. 156 41

Linker-insertion mutagenesis was used to isolate mutations in the Saccharomyces cerevisiae gene encoding the largest subunit of RNA polymerase II (RPO21, also called RPB1). The mutant rpo21 alleles carried on a plamid were introduced into a haploid yeast strain that conditionally expresses RPO21 from the inducible promoter pGAL10. Growth of this strain on medium containing glucose is sustained only if the plasmid-borne rpo21 allele encodes a functional protein. Of nineteen linker-insertion alleles tested, five (rpo21-4 to -8) were found that impose a temperature-sensitive (ts) lethal phenotype on yeast cells. Four of these five ts alleles encode mutant proteins in which the site of insertion lies near one of the regions of the largest subunit that have been conserved during evolution. Two of the ts mutants (rpo21-4 and rpo21-7) display pleiotropic phenotypes, including an auxotrophy for inositol and a decreased proliferation rate at the permissive temperature. The functional relationship between RPO21 and RPO26, the gene encoding the 17.9 kDa subunit shared by RNA polymerases I, II, and III was investigated by determining the ability of increased dosage of RPO26 to suppress the ts phenotype imposed by rpo21-4 to -8. Suppression of the ts defect was specific for the rpo21-4 allele and was accompanied by co-suppression of the inositol auxotrophy. These results suggest that mutations in the largest subunit of RNA polymerase II can have profound effects on the expression of specific subsets of genes, such as those involved in the metabolism of inositol. In the rpo21-4 mutant, these pleiotropic phenotypes can be attributed to a defective interaction between the largest subunit and the RPO26 subunit of RNA polymerase II.
Mol Gen Genet 1992 Apr
PMID:Isolation and phenotypic analysis of conditional-lethal, linker-insertion mutations in the gene encoding the largest subunit of RNA polymerase II in Saccharomyces cerevisiae. 158 9


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