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

Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2010; 107(23):10418-23. DOI: 10.1073/pnas.1000967107
Source: PubMed


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 lambdaP(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 lambdaP(R) promoter resemble the unstable, transcriptionally competent open complexes formed at ribosomal promoters.

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    • "The different lengths of the non-template strand might hinder the formation of the RNAP-non-template strand interactions that are required to progress into the open complex, and instead stall the complex in an RPi like state by mimicking different stages of DNA scrunching and/or promoter DNA engagement by RNAP (33,34). Burst and fast permanganate footprinting experiments showed that the RPo differs from the RPi by the position of the non-template strand in the active centre, allowing the assembly and DNA binding of the clamp and jaw domains (39). Transcription occurring in the RPi may limit the conversion to RPo by restricting necessary further conformational change in RNAP. "
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    ABSTRACT: The formation of the open promoter complex (RPo) in which the melted DNA containing the transcription start site is located at the RNA polymerase (RNAP) catalytic centre is an obligatory step in the transcription of DNA into RNA catalyzed by RNAP. In the RPo, an extensive network of interactions is established between DNA, RNAP and the σ-factor and the formation of functional RPo occurs via a series of transcriptional intermediates (collectively ‘RPi’). A single tryptophan is ideally positioned to directly engage with the flipped out base of the non-template strand at the +1 site. Evidence suggests that this tryptophan (i) is involved in either forward translocation or DNA scrunching and (ii) in σ54-regulated promoters limits the transcription activity of at least one intermediate complex (RPi) before the formation of a fully functional RPo. Limiting RPi activity may be important in preventing the premature synthesis of abortive transcripts, suggesting its involvement in a general mechanism driving the RPi to RPo transition for transcription initiation.
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    • "An alternative explanation for the inhibition effect observed in the presence of Gp2 is that in Eσ54 mutants the downstream DNA might fail to establish contacts with the downstream DNA-binding channel (dwDBC) that forms with the clamp, the jaw domain and other downstream mobile elements (22–24). Both, the correct positioning of the downstream DNA and the presence of a specific length of DNA for interaction with the jaw domain are crucial for the formation of productive open complexes (50). "
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    • "During the transcription, the RNAP using accessory factors binds to a specific DNA consensus sequence at the level of the promoter (P) forming a closed complex (RP c ). The RP c isomerises, through multiple intermediate states that can be promoter and factor specific [7] [8], to a final open complex state (RP o ), competent for transcription, where the double-stranded DNA has melted out and the transcription starting site (the +1 site) is at the active centre of RNAP. "
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