Yeast Transcript Elongation Factor (TFIIS), Structure and Function

Abstract

The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region. We have probed the function of this fragment of TFIIS using structureguided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix. TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action. The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation. Mutation of three aromatic residues in the zinc ribbon domain (Phe 269 , Phe 296 , and Phe 308 ) impaired both transcript cleavage and read-through. Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.Elongating RNA polymerase II stalls upon encountering blocks to elongation in vitro (1). In some cases, these transiently stalled polymerases convert to very stable arrested complexes. Arrested complexes are unable to resume transcription even after hours to weeks of incubation (2). The inability of such complexes to resume transcription results from a structural change in the stalled polymerase, which causes the active site to disengage from the 3Ј-end of the transcript (3). The general elongation factor TFIIS 1 reactivates arrested transcription complexes within minutes (4). The reactivation process involves a TFIIS-stimulated endonucleolytic cleavage of the transcript by the RNA polymerase II (5, 6), which relocates the polymerase active site to the new 3Ј-end of the RNA chain and allows for chain extension. The reactivation of stalled elongation complexes involves multiple steps, with the first being the interaction of TFIIS with RNA polymerase II. The TFIIS-binding domain on RNA polymerase II was identified by Friesen and colleagues (7), who discovered mutants in the largest subunit of RNA polymerase II, RPB1, that displayed the same phenotype as a strain deleted for the TFIIS gene (sensitivity to the drug 6-azauracil) and that also could be suppressed by overexpression of TFIIS. These mutants localized to a part of RPB1 between regions G and H, which are conserved from bacteria to man and are in close proximity to the RNA polymerase active site (8, 9). The genetic evidence for a TFIIS interacting domain was confirmed biochemically, when two of the mutant RNA polymerases were purified and shown directly to have 500-fold lower affinity for TFIIS compared with the wild-type polymerase (10).Transcript cleavage is the next essential step in the reactivation process. It is now clear that R...

Intrinsic Transcript Cleavage in Yeast RNA Polymerase II Elongation Complexes

“…2, lane 7) (see also Ref. 25). Two major cleavage products, C1 and C2, remain associated with active ternary complexes; the C1 cleavage product is generated prior to C2 (25).…”

“…A minimal sequence sufficient to block RNA polymerase II elongation complexes is boxed (27). C1 demarcates the position of yeast and mammalian ternary complexes after the initial transcript cleavage event from the T1a site (25,37). B, sequences used to prepare G135, U138, G143, G152, and C158 ternary complexes with the immobilized pCpRW106.…”

“…The T1a (ϳ200 nt), C1, and C2 transcripts (25) formed on pCpGEMTERM were resolved on 6% polyacrylamide (38% acrylamide:2% bisacrylamide) gels containing 7 M urea in 1ϫ TBE. RNA oligomers released during transcript cleavage were resolved on either 15% (38% acrylamide:2% bisacrylamide) or 23% (40% acrylamide:6% bisacrylamide) gels containing 7 M urea in 1ϫ TBE.…”

“…Both RPII and RPII⌬9 were purified as described previously (24,25), except that 10 M ZnCl 2 was included in all buffers. The polymerase preparations were stored at Ϫ80°C in 10 mM Tris-HCl, pH 7.9, 40 mM ammonium sulfate, 10% glycerol, 2.5 mM DTT, 100 mM EDTA, and 10 M ZnCl 2 .…”

“…The polymerase preparations were stored at Ϫ80°C in 10 mM Tris-HCl, pH 7.9, 40 mM ammonium sulfate, 10% glycerol, 2.5 mM DTT, 100 mM EDTA, and 10 M ZnCl 2 . Recombinant yeast TFIIS was purified as previously described (25).…”