Chen Bo-Shiun, Hampsey Michael
Division of Nucleic Acids Enzymology, Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
Mol Cell Biol. 2004 May;24(9):3983-91. doi: 10.1128/MCB.24.9.3983-3991.2004.
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-TFIIB 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.
通用转录因子TFIIB是精确起始所必需的，尽管RNA聚合酶II（RNAP II）识别起始位点的机制尚未完全清楚。在此，我们描述了对酵母TFIIB的一种改变形式进行遗传和生化分析的结果，该形式在“B-指”结构域中含有精氨酸78→半胱氨酸（R78C）替换。TFIIB R78C将起始位点选择向下游偏移至正常位点之外，并赋予冷敏感生长缺陷（Csm(-)）。对R78C Csm(-)表型的抑制确定了TFIIB与RNAP II的Rpb2亚基之间的功能相互作用，并定义了Rpb2在起始位点选择中的新作用。rpb2抑制子编码甘氨酸369→丝氨酸（G369S）替换，位于Rpb2的“叶”结构域且靠近之前被鉴定为起始位点选择效应器的Rpb9亚基。RNAP II的Rpb2-Rpb9“叶-颌”区域位于催化中心下游且远离RNAP II-TFIIB相互作用位点。TFIIB R78C突变体提取物在启动子特异性连续转录方面存在缺陷，但产生了改变的流产起始产物模式，表明R78C缺陷并不排除起始。sua7-3 rpb2-101双突变体在体内对6-氮杂尿嘧啶敏感，在体外对核苷三磷酸底物耗竭敏感。结合最近酵母RNAP II-TFIIB复合物的X射线结构，这些结果确定了TFIIB的B-指结构域与RNAP II的远端叶-颌区域之间的功能相互作用，并为起始位点选择机制提供了深入了解。
