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• W2072420783 abstract "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 λ P R promoter load this region of duplex DNA into the active site cleft of Escherichia coli RNAP, forming the closed, permanganate-unreactive intermediate I 1 . Conversion to the subsequent intermediate I 2 overcomes a large enthalpic barrier. Is I 2 open? Here we create a burst of I 2 by rapidly destabilizing open complexes (RP o ) with 1.1 M NaCl. Fast footprinting reveals that thymines at positions from -11 to +2 in I 2 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 I 2 to I 1 conversion rate determined by filter binding. In I 2 , permanganate reactivity of the +1 thymine on the template (t) strand is the same as the RP o control, whereas nontemplate (nt) thymines are significantly less reactive than in RP o . We propose that: ( i ) the +1(t) thymine is in the active site in I 2 ; ( ii ) conversion of I 2 to RP o 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 RP o . We hypothesize that unstable open intermediates at the λ P R promoter resemble the unstable, transcriptionally competent open complexes formed at ribosomal promoters." @default.
• W2072420783 created "2016-06-24" @default.
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• W2072420783 date "2010-05-18" @default.
• W2072420783 modified "2023-09-27" @default.
• W2072420783 title "One-step DNA melting in the RNA polymerase cleft opens the initiation bubble to form an unstable open complex" @default.
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• W2072420783 doi "https://doi.org/10.1073/pnas.1000967107" @default.
• W2072420783 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2890804" @default.
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