Transcription Elongation Factor S-II Confers Yeast Resistance to 6-Azauracil by Enhancing Expression of the SSM1 Gene

Abstract

Loss of function of S-II makes yeast sensitive to 6-azauracil. Here, we identified a multi-copy suppressor gene of this phenotype, termed SSM1 (suppressor of 6-azauracil sensitivity of the S-II null mutant 1), that encodes a novel protein consisting of 280 amino acid residues. Although both the SSM1 null mutant and the S-II/SSM1 double null mutant were viable under normal growth conditions, they resembled the S-II null mutant in being sensitive to 6-azauracil. Expression of the SSM1 gene was found to be repressed in the S-II null mutant but was restored by overexpression of chimeric S-II molecules that were able to stimulate transcription elongation by RNA polymerase II in vitro. Furthermore, we identified two transcription arrest sites within the transcription unit of the SSM1 gene in vitro that could be relieved by S-II. These results indicate that S-II confers yeast resistance to 6-azauracil by stimulating transcription elongation of the SSM1 gene.Transcription is a complex process controlled at various steps, such as initiation, elongation, and termination (1-4). Recently, it has become evident that expression of several cellular and virus genes is regulated at the transcription elongation step (5-8). So far, multiple transcription elongation factors, such as S-II (TFIIS) (9, 10), elongin (SIII) (11, 12), TFIIF (13-15), 17), and ELL (18), have been identified. Among them, S-II was originally purified and characterized from mouse Ehrlich ascites tumor cells as a specific stimulatory protein of RNA polymerase II (9, 10, 19 -21). Subsequently, S-II has been identified in various organisms and shown to make RNA polymerase II read-through intrinsic blocks within the transcription units of eukaryotic genes, in vitro, by promoting cleavage of the 3Ј-end of the nascent RNA by RNA polymerase II (22-32).To investigate the cellular functions of S-II in eukaryotic transcription, we have been studying S-II from yeast (Saccharomyces cerevisiae) (33-35). The yeast S-II null mutant is viable but becomes sensitive to 6-azauracil (6-AU) 1 (34, 36). By creating various deletion mutants of S-II, we found that the C-terminal 147 amino acid residues are sufficient for the stimulation of RNA polymerase II and suppression of 6-AU sensitivity (34). Furthermore, by creating chimeric molecules of mouse and yeast S-II, we found that the region between Pro-131 and Phe-270 is responsible for the species-specific interaction of S-II and RNA polymerase II (35). These results suggested that the 6-AU sensitivity of the S-II null mutant is caused by loss of function of S-II as a transcription elongation factor. However, the target gene(s) for S-II that confers yeast resistance to 6-AU has not yet been identified. To gain more insight into the role of S-II in the sensitivity of yeast to 6-AU, we have identified a gene, SSM1, that suppresses sensitivity to 6-AU. We found that S-II enhances transcription of the SSM1 gene, resulting in suppression of the sensitivity of the S-II null mutant to 6-AU. MATERIALS AND METHODS Isolation of Clones Suppre...

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

“…Presumably, a gene's transcription level in this case would be related to the number or location of pause or arrest site sequences within it. Until recently, no arrest sites have been described in yeast gene sequences in vitro nor shown to operate in vivo (22 , and DY108(rpb2-10 dst1) cells treated with 6AU (75 g/ml) for 30 min before galactose induction. RNA was prepared at the indicated times, and the blot was probed for GAL1 mRNA.…”

“…SSM1 expression is reduced in DST1 null yeast and is restored by SII expression (22). Specific sequences in SSM1 that arrest transcription in vitro could explain why this gene requires SII for its transcription (22). A pressing question is whether elongation factors contribute to the efficient transcription of many or most genes or if they are particularly important for the function of those genes with specific arrest sites.…”

“…Recently, a gene called SSM1 (suppressor of 6-azauracil sensitivity of the SII null mutant 1) was identified in a high copy suppressor screen of the 6AU s phenotype of a DST1 deletant (22). SSM1 expression is reduced in DST1 null yeast and is restored by SII expression (22). Specific sequences in SSM1 that arrest transcription in vitro could explain why this gene requires SII for its transcription (22).…”

“…Cumulatively, these results suggest that one of the hallmarks of mutation in elongation factor genes is a slow transcriptional induction phenotype and a reduction in the efficiency of mRNA synthesis. Recently, a gene called SSM1 (suppressor of 6-azauracil sensitivity of the SII null mutant 1) was identified in a high copy suppressor screen of the 6AU s phenotype of a DST1 deletant (22). SSM1 expression is reduced in DST1 null yeast and is restored by SII expression (22).…”

Use of an in Vivo Reporter Assay to Test for Transcriptional and Translational Fidelity in Yeast

“…Cells lacking SII are defective in transcriptional induction of a number of genes (10,12,35). To ensure that the luciferase activity determinations were not biased by a difference in luciferase mRNA levels between the DST1 deletant and cells wild-type for DST1, we measured the amount of transcript by Northern blotting (data not shown).…”