TY - JOUR
T1 - Functional Interaction between TFIIB and the Rpb2 Subunit of RNA Polymerase II
T2 - Implications for the Mechanism of Transcription Initiation
AU - Chen, Bo Shiun
AU - Hampsey, Michael
PY - 2004/5
Y1 - 2004/5
N2 - The general transcription factor TFIIB is required for accurate initiation, although the mechanism by which RNA polymerase II (RNAP II) identifies initiation sites is not well understood. Here we describe results from genetic and biochemical analyses of an altered form of yeast TFIIB containing an arginine-78 → cysteine (R78C) replacement in the "B-finger" domain. TFIIB R78C shifts start site selection downstream of normal and confers a cold-sensitive growth defect (Csm-). Suppression of the R78C Csm- phenotype identified a functional interaction between TFIIB and the Rpb2 subunit of RNAP II and defined a novel role for Rpb2 in start site selection. The rpb2 suppressor encodes a glycine-369 → serine (G369S) replacement, located in the "lobe" domain of Rpb2 and near the Rpb9 subunit, which was identified previously as an effector of start site selection. The Rpb2-Rpb9 "lobe-jaw" region of RNAP II is downstream of the catalytic center and distal to the site of RNAP II-TFIIB interaction. A TFIIB R78C mutant extract was defective for promoter-specific run-on transcription but yielded an altered pattern of abortive initiation products, indicating that the R78C defect does not preclude initiation. The sua7-3 rpb2-101 double mutant was sensitive to 6-azauracil in vivo and to nucleoside triphosphate substrate depletion in vitro. In the context of the recent X-ray structure of the yeast RNAP II-TFIIP complex, these results define a functional interaction between the B-finger domain of TFIIB and the distal lobe-jaw region of RNAP II and provide insight into the mechanism of start site selection.
AB - The general transcription factor TFIIB is required for accurate initiation, although the mechanism by which RNA polymerase II (RNAP II) identifies initiation sites is not well understood. Here we describe results from genetic and biochemical analyses of an altered form of yeast TFIIB containing an arginine-78 → cysteine (R78C) replacement in the "B-finger" domain. TFIIB R78C shifts start site selection downstream of normal and confers a cold-sensitive growth defect (Csm-). Suppression of the R78C Csm- phenotype identified a functional interaction between TFIIB and the Rpb2 subunit of RNAP II and defined a novel role for Rpb2 in start site selection. The rpb2 suppressor encodes a glycine-369 → serine (G369S) replacement, located in the "lobe" domain of Rpb2 and near the Rpb9 subunit, which was identified previously as an effector of start site selection. The Rpb2-Rpb9 "lobe-jaw" region of RNAP II is downstream of the catalytic center and distal to the site of RNAP II-TFIIB interaction. A TFIIB R78C mutant extract was defective for promoter-specific run-on transcription but yielded an altered pattern of abortive initiation products, indicating that the R78C defect does not preclude initiation. The sua7-3 rpb2-101 double mutant was sensitive to 6-azauracil in vivo and to nucleoside triphosphate substrate depletion in vitro. In the context of the recent X-ray structure of the yeast RNAP II-TFIIP complex, these results define a functional interaction between the B-finger domain of TFIIB and the distal lobe-jaw region of RNAP II and provide insight into the mechanism of start site selection.
UR - http://www.scopus.com/inward/record.url?scp=1942422184&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=1942422184&partnerID=8YFLogxK
U2 - 10.1128/MCB.24.9.3983-3991.2004
DO - 10.1128/MCB.24.9.3983-3991.2004
M3 - Article
C2 - 15082791
AN - SCOPUS:1942422184
SN - 0270-7306
VL - 24
SP - 3983
EP - 3991
JO - Molecular and Cellular Biology
JF - Molecular and Cellular Biology
IS - 9
ER -