Promoter specificity determinants of T7 RNA polymerase
Promoter specificity determinants of T7 RNA polymerase
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DOI：
10.1073/pnas.95.2.515
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发表时间：
1998-01-20
影响因子：
11.1
通讯作者：
Durbin, RK
中科院分区：
文献类型：
--
作者：
Rong, MQ;He, B;Durbin, RK
T7 RNA聚合酶（RNAP）对其启动子序列的高度特异性部分由伸入DNA结合裂缝的特异性环（残基742-773）介导（1），先前的工作证明了该环中第748位氨基酸残基（N748）在区分第-10和-11位碱基对（bp）中的作用（2）。其他噬菌体RNAP和它们的启动子的序列的比较表明，额外的接触，可能是重要的启动子识别。我们已经发现，改变T7 RNAP中758位的氨基酸残基导致酶对-8位bp的特异性改变。N748的bp在-10和-11处，Q758的bp在-8处）提供了关于特异性环相对于启动子上游区的布置的信息。结果表明，大量的重排的环（和/或DNA）可能需要允许这些氨基酸在启动子识别过程中与它们的同源碱基对相互作用。
The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742-773) that projects into the DNA binding cleft (1), Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions -10 and -11 (2). A comparison of the sequences of other phage RNAPs and their promoters suggested additional contacts that might be important in promoter recognition. We have found that changing the amino acid residue at position 758 in T7 RNAP results in an enzyme with altered specificity for the bp at position -8, The identification of two amino acid:base pair contacts (i.e., N748 with the bp at -10 and -11, and Q758 with the bp at -8) provides information concerning the disposition of the specificity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and/or the DNA) are likely to be required to allow these amino acids to interact with their cognate base pairs during promoter recognition.