Raskin C A, Diaz G, Joho K, McAllister W T
Morse Institute for Molecular Genetics Department of Microbiology and Immunology State University of New York, Brooklyn 11203-2098.
J Mol Biol. 1992 Nov 20;228(2):506-15. doi: 10.1016/0022-2836(92)90838-b.
The bacteriophage T3 and T7 RNA polymerases (RNAP) are closely related, yet exhibit high specificity for their own promoter sequences. In this work the primary determinant of T7 versus T3 promoter specificity has been localized to a single amino acid residue at position 748 in the T7 RNAP. Substitution of this residue (Asn) with the corresponding residue found in T3 RNAP (Asp) results in a switch in promoter specificity, and specifically alters recognition of the base pairs (bp) at positions -11 and, possibly, -10 in the promoter. A complementary mutation in T3 RNAP (T3-D749N) results in a similar switch in promoter preference for that enzyme. The hierarchy of bp preference by the mutant and wild-type enzymes for bp at -10 and -11, and the results of previous experiments, lead to a model for specificity in which it is proposed that N748 in T7 RNAP (and D749 in T3 RNAP) make specific hydrogen bonds with bases at -11 and -10 on the non-template strand in the major groove. The specificity determining region of T7 RNAP does not appear to exhibit homology to any known sequence-dependent DNA binding motif.
噬菌体T3和T7 RNA聚合酶（RNAP）密切相关，但对其自身的启动子序列表现出高度特异性。在这项研究中，T7与T3启动子特异性的主要决定因素已定位到T7 RNAP中第748位的单个氨基酸残基。将该残基（天冬酰胺）替换为T3 RNAP中相应的残基（天冬氨酸）会导致启动子特异性的转换，并特别改变启动子中-11位以及可能的-10位碱基对（bp）的识别。T3 RNAP中的互补突变（T3-D749N）导致该酶的启动子偏好发生类似转换。突变型和野生型酶对-10和-11位碱基对的碱基偏好层次以及先前实验的结果，得出了一个特异性模型，其中提出T7 RNAP中的N748（和T3 RNAP中的D749）在大沟中与非模板链上-11和-10位的碱基形成特定的氢键。T7 RNAP的特异性决定区域似乎与任何已知的序列依赖性DNA结合基序均无同源性。
