Identification of the Region in Yeast S-II That Defines Species Specificity in Its Interaction with RNA Polymerase II

Abstract

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 ...

The Rpb6 Subunit of Fission Yeast RNA Polymerase II Is a Contact Target of the Transcription Elongation Factor TFIIS

“…The TFIIS of S. cerevisiae is composed of three domains, I, II, and III; the nuclear magnetic resonance structures have been solved for the C-terminal proximal domains II and III (29). Domains II and III are known to be essential for interaction with the RNA polymerase II (1,7,39). S. cerevisiae mutants carrying mutations in the PPR2 gene for TFIIS have been isolated; these mutants show high-level sensitivity to 6AU that inhibits IMP dehydrogenase and ultimately results in limitation in GTP and UTP pools (11).…”

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

“…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.…”

“…To investigate the cellular functions of S-II in eukaryotic transcription, we have been studying S-II from yeast (Saccharomyces cerevisiae) (33)(34)(35). The yeast S-II null mutant is viable but becomes sensitive to 6-azauracil (6-AU) 1 (34,36).…”

Stimulation of RNA polymerase II transcript cleavage activity contributes to maintain transcriptional fidelity in yeast

“…Domain I, located in the N-terminal region, is involved in interactions with other transcription factors (Saso et al 2003;Wery et al 2004). The other two regions, domain II and domain III, are essential for binding to RNA polymerase II and for both the cleavage and readthrough stimulation activities, respectively (Nakanishi et al 1995;Shimoaraiso et al 1997;Awrey et al 1998). X-ray structure analysis of the yeast RNA polymerase II-S-II complex revealed that domain III of S-II intrudes into the pore of RNA polymerase II and approaches the catalytic center of RNA polymerase II (Kettenberger et al 2003).…”