RNA polymerase II mutants defective in transcription of a subset of genes.

Abstract

Saccharomyces cerevisiae RNA polymerase II conditional mutants that selectively disrupt the synthesis of specific mRNAs were isolated. At the permissive temperature, several of the mutants were inositol auxotrophs as a result of inadequate induction of INO1 transcription. The transcriptional defects exhibited by one of these Ino-mutants (rpb2-2) were further investigated. The induction of GAL1O and HIS4 transcription in rpb2-2 strains was similar to that of wild-type strains, in contrast to the lack of induction of INOI transcription. When shifted to the nonpermissive temperature, cells containing rpb2-2 continued to accumulate some mRNAs but not others. Together, these results indicate that transcription of specific genes can be disrupted by RNA polymerase II mutations. The rpb2-2 allele alters an amino acid residue that occurs in a highly conserved segment of the RPB2 protein and that is shared by homologous subunits in other species.The regulation of eucaryotic gene expression occurs primarily at the level of selective transcription initiation by RNA polymerase II. Specific promoter sequences, transcriptional activating protein factors, and RNA polymerase all play a role in transcription initiation. Transcriptional activating proteins bind specifically to cis-acting promoter elements such as enhancer sequences or the TATA box (12, 21). The most thoroughly studied transcriptional activating proteins have separate domains for DNA-binding and transcriptional activation (reviewed in references 13 and 22). The mechanism(s) by which these proteins activate transcription initiation by RNA polymerase II is unclear.RNA polymerase II is composed of 9 to 14 polypeptides of 10 to 220 kilodaltons that are substantially conserved in size and structure throughout eucaryotes (31). The yeast nuclear RNA polymerases are among the best studied of the eucaryotic polymerases. Yeast RNA polymerase II contains 10 polypeptides, 7 of which are unique to the enzyme and 3 of which are shared with the other two polymerases. Genes encoding seven of the yeast RNA polymerase II subunits have been isolated and characterized (17,18,29,(35)(36)(37); N. A. Woychik, S.-M. Liao, P. Kolodziej, and R. A. Young, Genes Dev., in press). The sequences of the two largest subunits, RPBJ and RPB2 (1,35), reveal that the RPB1 and RPB2 proteins are similar to the Escherichia coli RNA polymerase subunits P' (RpoC) and 1 (RpoB), respectively.Biochemical and genetic evidence indicates that the RNA polymerase ,B subunit interacts with the a subunit, which is essential for selective transcription initiation in E. coli (9,26,32,38). It is not clear how components of the eucaryotic RNA polymerase interact with transcription factors.To obtain clues to the functions of the RPBI and RPB2 subunits, we have isolated and investigated RNA polymerase mutants that affect mRNA synthesis. We previously described a temperature-sensitive RPBJ mutant that rapidly ceases all mRNA synthesis upon transfer to the nonpermis-

Analysis of Gene Induction and Arrest Site Transcription in Yeast with Mutations in the Transcription Elongation Machinery

“…The pol II mutation rpb2-10, which changes a conserved proline near the active site to a serine and generates an arrest-prone enzyme that transcribes slowly in vitro, confers a similar "slow-induction" phenotype upon yeast as do the ELP1, ELP3, and DST1 deletions. The original description of this allele included inositol auxotrophy and reduced levels of INO1 transcription (21). Combining these two mutations (rpb2-10 and dst1) consistently resulted in a more severe defect than either alone, in agreement with earlier findings that the double mutant displayed synthetic 6AU hypersensitivity, contained less poly(A) ϩ mRNA, and was more severely compromised in its ability to induce transcription of IMD2 (PUR5), than either single mutant (5,12).…”

“…These cells are compromised in their ability to induce transcription of the gene for IMP dehydrogenase, IMD2 (also known as PUR5) in response to 6AU treatment (12). As well, the rpb2-10 mutation results in cold sensitivity and inositol auxotrophy, the latter phenotype is attributed to the inability to transcribe INO1 (21). Cells lacking functional SII and containing the slow elongation mutation display a synergistic sensitivity to 6AU.…”

Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

“…Hence, 6AU sensitivity has been exploited as a bioassay for transcription elongation. Indeed, mutations in two of the subunits of RNA PolII yield 6AU-sensitive yeast (3,25,34,35). Some of these mutations generate enzymes that are defective in elongation in vitro, either by reducing the affinity of SII for polymerase (45) or by yielding an arrest-prone enzyme with a low elongation rate (25).…”

“…Nevertheless, direct evidence that SII acts as an elongation factor in vivo is lacking, and the effect of mutations in DST1 or 6AU treatment on mRNA levels has not been explored. Little is known about target genes that are particularly reliant upon SII for their transcription or whose transcription rate is sensitive to a compromised elongation machinery.rpb2-4 (A1016T) and rpb2-10 (P1018S) are two point mutations in the gene encoding the second largest subunit of PolII (RPB2) that confer 6AU sensitivity upon yeast (25,34,35). The rpb2-10 mutation yields polymerases which possess an abnormally low rate of elongation in vitro and a higher propensity to become arrested (25).…”

Trans‐dominant mutations in the 3′‐terminal region of the rpoB gene define highly conserved, essential residues in the β subunit of RNA polymerase: the GEME motif

“…The G→D substitution of the highly conserved residue (equivalent to G1282 in E. coli ␤; Fig. 2) was found to result in defective transcription of certain genes (Scafe et al 1990b).…”