YEAST TRANSCRIPT ELONGATION-FACTOR (TFIIS), STRUCTURE AND FUNCTION

The transcriptionally active fragment of the yeast RNA polymerase II t ranscription 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 structure-guided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinit y 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 TFI IS 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 zi nc ribbon domain (Phe(269), Phe(296), and Phe(308)) impaired both tran script cleavage and read-through. Mutations introduced in the linker r egion between residues 240 and 245 and between 250 and 255 also severe ly impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a cr itical structure in the context of the elongation complex.