The yeast S-II null mutant is viable, but the mutation induces sensitivity to 6-azauracil. To examine whether the region needed for stimulation of RNA polymerase II and that for suppression of 6-azauracil sensitivity in the S-II molecule could be separated, we constructed various deletion mutants of S-II and expressed them in the null mutant using the GAL1 promoter to see if the mutant proteins suppressed 6-azauracil sensitivity. We also expressed these constructs in Escherichia coli, purified the mutant proteins to homogeneity, and examined if they stimulated RNA polymerase II. We found that a mutant protein lacking the first 147 amino acid residues suppressed 6-azauracil sensitivity but that removal of 2 additional residues completely abolished the suppression. A mutant protein lacking the first 141 residues had activity to stimulate RNA polymerase II, whereas removal of 10 additional residues completely abolished this activity. We also examined arrest-relief activity of these mutant proteins and found that there is a good correlation between RNA polymerase II-stimulating activity and arrest-relief activity. Therefore, at least the last 168 residues of S-II are sufficient for expressing these three activities.

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...

Yeast S-II was found to stimulate yeast RNA polymerase II only and not mouse RNA polymerase II. To identify the molecular region of S-II that defines species specificity, we constructed six hybrid S-II molecules consisting of three regions from yeast and/or Ehrlich cell S-II and examined their activity in terms of RNA polymerase II specificity and suppression of 6-azauracil sensitivity in the yeast S-II null mutant. We found that the region 132-270 (amino acid positions) of yeast S-II is indispensable for specific interaction with yeast RNA polymerase II in vitro and for suppression of 6-azauracil sensitivity in vivo. The corresponding region of Ehrlich cell S-II, the region 132-262, was also shown to be essential for its interaction with mouse RNA polymerase II. This region is known to be less conserved than the Nand C-terminal regions in the S-II family suggesting that it is important in the interaction with transcription machinery proteins in a tissue and/or species-specific manner.Transcription initiation is a complex process that involves protein-protein and protein-nucleic acid interactions, and factors participating in this process have been extensively characterized (1-4). On the other hand, regulation of gene expression at the transcription elongation level has been less thoroughly studied. Recently, it has become evident that various cellular and viral genes are regulated at the level of transcription elongation (5-8). Thus, transcription elongation is likely to also be a crucial step for eukaryotic gene expression that involves various transcription elongation factors such as S-II (TFIIS) (9, 10), Elongin (SIII) (11, 12), TFIIF (13-15), and ELL (16).Transcription elongation factor S-II was originally purified from mouse Ehrlich ascites tumor cells as a specific stimulatory protein of RNA polymerase II, and it was thought to participate in eukaryotic transcription (9,10,(17)(18)(19). Subsequently, S-II was purified from various organisms and was shown to enable RNA polymerase II to read through blocks of transcription elongation within the transcription units of many eukaryotic genes by promoting cleavage of the 3Ј-end of the nascent RNA by RNA polymerase .Previously, we purified and characterized S-II from Saccharomyces cerevisiae (31, 32). Yeast S-II was found to contain an N-terminal region of 73 residues and a C-terminal region, including a zinc ribbon motif, that are relatively well conserved in the S-II proteins of many other species (33,34). A gene disruption experiment revealed that the S-II null mutant is viable but becomes sensitive to 6-azauracil.During the study of yeast S-II, we found that there is a strict species specificity in the combination of S-II and RNA polymerase II. Yeast S-II did not stimulate mouse RNA polymerase II and vice versa. To identify the region of the S-II molecule that prescribes species specificity, we constructed various hybrid clones between yeast and Ehrlich cell S-II. We found that the region between Pro-131 and Phe-270 is needed for yeast S-II to interact with ...