Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

Abstract

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 (RP021). A screen for mutations in RP021 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 rpo2l 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 rpo2l 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 rpo2l 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.Eukaryotic RNA polymerases (RNAPs) are highly conserved, multisubunit enzymes composed of two large subunits associated with several smaller polypeptides, some of which are shared by all three RNAPs or are common to only RNAPI and RNAPIII (reviewed in references 68, 73, and 83). In addition to RNAPII, mRNA synthesis requires several general factors for the assembly of initiation complexes at promoters and subsequently the stimulation of transcript elongation and termination (reviewed in references 66 and 68). Although gene expression is most often regulated at the level of transcription initiation in eukaryotes, a growing number of studies indicate that control of gene expression also occurs at the level of transcript elongation (reviewed in references 37 and 79). In eukaryotes and their viruses, examples of genes known to be regulated at the level of transcript elongation include the human immunodeficiency virus type 1 long terminal repeat (60), the simian virus 40 late-transcription unit (4), the major late-transcription unit of adenovirus (44), the proto-oncogenes c-myc (8), c-myb (6), c-mos (48), c-fos (15, 49), and L-myc (41), as well as the adenosine deaminase gene (13). In Drosophila melanogaster, work performed by Gilmour and Lis (26) identified, little is known about the factors that may be involved in this process. Clear...

Evolution, biogenesis and function of promoter-associated RNAs

“…8 Intriguingly, TFIIS is not essential for yeast viability 44,45 and plants defective in TFIIS show only a mild reduced seed dormancy phenotype, 46 whereas homozygous null mutant TFIIS mice die at midgestation. 47 This may reflect an important role for TFIIS in gene regulation through tiRNA biogenesis in developmentally complex animals.…”

Selectivity and proofreading both contribute significantly to the fidelity of RNA polymerase III transcription

“…PPR2 is not essential for yeast viability (38). Rpb9 subunit of Pol II, a C11 paralogue recently identified as important for the in vivo fidelity of Pol II and suggested to act in proofreading (34), is also encoded by a nonessential gene.…”

Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces cerevisiae

“…This activity is believed to assist RNAP II in overcoming blocks to elongation by repositioning the 3Ј end of the nascent mRNA in the active site of arrested transcription complexes (Reines et al 1992(Reines et al , 1993Christie et al 1994). The lack of TFIIS renders cells hypersensitive to 6AU and is lethal in combination with mutations in RPB2 (Archambault et al 1992). We tested several different dyn2 reporters in strains expressing different levels of TFIIS.…”

“…Between 7.5 and 15 fmole of each 5Ј-32 P-labeled primer was annealed to 5 to 11 µg total RNA (5 min at 65°C followed by 25 min at 42°C). Extension reactions were performed at 42°C as previously described (Ares PPR2 gene inserted into pFL44D Archambault et al 1992 Transcription and exon skipping www.rnajournal.org and Igel 1990) using either AMV RT (Life Sciences) or M-MLV RT (GIBCO; Fig. 6B) in the buffer suggested by the manufacturers.…”