Yeast transcript elongation factor (TFIIS), structure and function. II: RNA polymerase binding, transcript cleavage, and read-through.
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 structure-guided 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 (Phe269, Phe296, and Phe308) 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.
C.H. Best Institute, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
D E
DE
Awrey
N
N
Shimasaki
C
C
Koth
R
R
Weilbaecher
V
V
Olmsted
S
S
Kazanis
X
X
Shan
J
J
Arellano
C H
CH
Arrowsmith
C M
CM
Kane
A M
AM
Edwards
D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; D015032:Zinc / Q000737:chemistry
D012333:RNA, Messenger; D014157:Transcription Factors; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D012319:RNA Polymerase II; D015032:Zinc