Abstract
Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ(A) dissociation.
Highlights
Transcription initiation is a major control point of gene expression
The architecture of EσA recognition of the key −35 and −10 promoter elements was delineated by the structure of Thermus aquaticus (Taq) EσA bound to an upstream fork promoter fragment, but the low resolution (6.5 A ) prevented the visualization of molecular details (Murakami et al, 2002b)
We formed a complete RPo by combining Taq EΔ1.1σA with a duplex promoter DNA scaffold (−36 to +12 with respect to the transcription start site at +1) but with a non-complementary transcription bubble generated by altering the sequence of the t-strand DNA from −11 to +2
Summary
Transcription initiation is a major control point of gene expression. The initiation process is best understood in the bacterial system (Saecker et al, 2011) where the conserved ∼400 kD catalytic core of the RNA polymerase (RNAP or E, subunit composition α2ββ′ω) combines with the promoter-specificity factor σA to form the holoenzyme (EσA), which locates promoter DNA and unwinds 12–14 base pairs (bps) of the DNA duplex to yield the transcription-competent open promoter complex (RPo). Before transitioning to a stable elongation complex, steric clash between the elongating RNA transcript and elements of σ set up abortive initiation, where the RNAP repeatedly generates and releases short transcripts without dissociating from the promoter (McClure et al, 1978; Murakami et al, 2002a; Goldman et al, 2009). High resolution crystal structures defined key, sequence-specific interactions of σ with the −35 element (Campbell et al, 2002), the melted −10 element (Feklistov and Darst, 2011), as well as with downstream promoter DNA in the context of holoenzyme (Zhang et al, 2012), these structures did not contain the full transcription bubble with the upstream double-strand/single-strand (ds/ss) DNA junction at the upstream edge of the −10 element where transcription bubble formation initiates
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