期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2010
卷号：107
期号：23
页码：10418-10423
DOI：10.1073/pnas.1000967107
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Though opening of the start site (+1) region of promoter DNA is required for transcription by RNA polymerase (RNAP), surprisingly little is known about how and when this occurs in the mechanism. Early events at the{lambda} PR promoter load this region of duplex DNA into the active site cleft of Escherichia coli RNAP, forming the closed, permanganate-unreactive intermediate I1. Conversion to the subsequent intermediate I2 overcomes a large enthalpic barrier. Is I2 open? Here we create a burst of I2 by rapidly destabilizing open complexes (RPo) with 1.1 M NaCl. Fast footprinting reveals that thymines at positions from -11 to +2 in I2 are permanganate-reactive, demonstrating that RNAP opens the entire initiation bubble in the cleft in a single step. Rates of decay of all observed thymine reactivities are the same as the I2 to I1 conversion rate determined by filter binding. In I2, permanganate reactivity of the +1 thymine on the template (t) strand is the same as the RPo control, whereas nontemplate (nt) thymines are significantly less reactive than in RPo. We propose that: (i) the +1(t) thymine is in the active site in I2; (ii) conversion of I2 to RPo repositions the nt strand in the cleft; and (iii) movements of the nt strand are coupled to the assembly and DNA binding of the downstream clamp and jaw that occurs after DNA opening and stabilizes RPo. We hypothesize that unstable open intermediates at the{lambda} PR promoter resemble the unstable, transcriptionally competent open complexes formed at ribosomal promoters.
