Abstract
Critical contacts made between the RNA polymerase (RNAP) holoenzyme and promoter DNA modulate not only the strength of promoter binding, but also the frequency and timing of promoter escape during transcription. Here, we describe a single-molecule optical-trapping assay to study transcription initiation in real time, and use it to map contacts formed between σ70 RNAP holoenzyme from E. coli and the T7A1 promoter, as well as to observe the remodeling of those contacts during the transition to the elongation phase. The strong binding contacts identified in certain well-known promoter regions, such as the −35 and −10 elements, do not necessarily coincide with the most highly conserved portions of these sequences. Strong contacts formed within the spacer region (−10 to −35) and with the −10 element are essential for initiation and promoter escape, respectively, and the holoenzyme releases contacts with promoter elements in a non-sequential fashion during escape.
Highlights
Critical contacts made between the RNA polymerase (RNAP) holoenzyme and promoter DNA modulate the strength of promoter binding, and the frequency and timing of promoter escape during transcription
We find that a strong contact within the so-called “spacer region” of the promoter, situated between the well characterized –10 and –35 elements, is essential to the initiation process, and that the RNAP holoenzyme releases its contacts with various promoter elements in a non-sequential order during promoter escape
The assay consists of two polystyrene beads, each held in a separate optical trap[26], and attached to double-stranded DNA (dsDNA) handles flanking a single DNA hairpin that carries a promoter with a transcription initiation site (Fig. 1a)
Summary
Critical contacts made between the RNA polymerase (RNAP) holoenzyme and promoter DNA modulate the strength of promoter binding, and the frequency and timing of promoter escape during transcription. We describe a single-molecule optical-trapping assay to study transcription initiation in real time, and use it to map contacts formed between σ70 RNAP holoenzyme from E. coli and the T7A1 promoter, as well as to observe the remodeling of those contacts during the transition to the elongation phase. The strong binding contacts identified in certain well-known promoter regions, such as the −35 and −10 elements, do not necessarily coincide with the most highly conserved portions of these sequences. Contacts mediated between promoter elements and the RNAP holoenzyme modulate the frequency of transcription initiation, and thereby regulate gene expression[12]. How does RNAP remodel its contacts with the promoter DNA during the initiation phase, leading to the formation of the EC?
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