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...

TFIIS is a general transcription elongation factor that helps arrested RNA polymerase II elongation complexes resume transcription. We have previously shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III). The three-dimensional structures of domain II and part of domain III have been previously reported, but neither domain can autonomously stimulate transcription elongation. Here we report the NMR structural analysis of residues 131-309 of yTFIIS which retains full activity and contains all of domains II and III. We confirm that the structure of domain II in the context of fully active yTFIIS is the same as that determined previously for a shorter construct. We have determined the structure of the C-terminal zinc ribbon domain of active yTFIIS and shown that it is similar to that reported for a shorter construct of human TFIIS. The region linking domain II with the zinc ribbon of domain III appears to be conformationally flexible and does not adopt a single defined tertiary structure. NMR analysis of inactive mutants of yTFIIS support a role for the linker region in interactions with the transcription elongation complex.The rate of transcript elongation by RNA polymerase II is regulated by elongation factors, of which there are two classes with different mechanisms of action. One class, which includes elongin, ELL, pTEFb, and RAP74 stimulates the rate of nucleotide incorporation. The other class, includes TFIIS, its viral and archael sequence homologues, and the bacterial proteins, greA and greB. These proteins do not affect the rate of nucleotide incorporation, but rather stimulate an activity in RNA polymerase II that enables it to transcribe throught blocks to elongation such as DNA-binding proteins, DNA-binding drugs, or particular sequences of DNA that promote transcription arrest (1).Although TFIIS 1 and its bacterial homologues have very similar mechanisms of action, their primary sequences and three-dimensional structures are quite different. GreA and greB each comprise 160 amino acids that form two 80-residue structural domains. In greA, the N-terminal domain is composed of two extended, coiled antiparallel ␣-helices, and can be cross-linked to the nascent RNA when bound to RNA polymerase in a stalled elongation complex (2). The C-terminal domain of greA is globular and is composed of a ␤-sheet that cradles an ␣-helix. GreB is a sequence homologue of greA and likely has an identical tertiary structure. yTFIIS is composed of three domains (domains I, II, and III) as defined by limited proteolysis and structural studies using nuclear magnetic resonance (NMR) spectroscopy (3) (see Fig. 1). Domains II and III, which together extend from residues 131-309 in yeast, are sufficient for transcription activity. Domain II (residues 131-240 in yeast) contains a three-helix bundle (3) and domain III is a zinc ribbon, which contains a three-stranded ␤-sheet, stabilized by a tetrad of cysteine residues that chelate a zinc ion (4). Domain II is conserved in TFIIS homologues and is postulated to int...