Awrey D E, Shimasaki N, Koth C, Weilbaecher R, Olmsted V, Kazanis S, Shan X, Arellano J, Arrowsmith C H, Kane C M, Edwards A M
C.H. Best Institute, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
J Biol Chem. 1998 Aug 28;273(35):22595-605. doi: 10.1074/jbc.273.35.22595.
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.
酵母RNA聚合酶II转录延伸因子TFIIS的转录活性片段由一个三螺旋束和一个通过连接区相连的锌带基序组成。我们利用结构导向诱变技术探究了TFIIS这一片段的功能。螺旋束结构域与RNA聚合酶II结合的亲和力与全长TFIIS相同，这种相互作用由第三螺旋外表面的一个碱性区域介导。无法与RNA聚合酶II结合的TFIIS突变体在转录活性方面无活性，这证实了聚合酶结合在TFIIS作用机制中的核心作用。连接区和锌带区在促进新生转录本的切割以及越过延伸阻碍进行通读方面发挥作用。锌带结构域中三个芳香族残基（苯丙氨酸269、苯丙氨酸296和苯丙氨酸308）的突变损害了转录本切割和通读。在残基240和245之间以及250和255之间的连接区引入的突变也严重损害了转录本切割和通读活性。我们的分析表明，TFIIS的连接区在延伸复合物的背景下可能采用了一种关键结构。
