One-step DNA melting in the RNA polymerase cleft opens the initiation bubble to form an unstable open complex

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 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? Herewecreate a burst of I-2 by rapidly destabilizing opencomplexes (RPo) with 1.1MNaCl. Fast footprinting reveals that thymines at positions from -11 to+2 inI(2) arepermanganate-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, permanganatereactivity 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 I-2; (ii) conversion of I-2 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.PR promoter resemble the unstable, transcriptionally competent open complexes formed at ribosomal promoters.