Abstract
Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA. Despite extensive biochemical and genetic analyses, puzzle remains, in part, because of a lack of precise structural information for σ appropriation complex. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact σ appropriation complex, comprising AsiA, MotA, Escherichia coli RNA polymerase (RNAP), σ70 and a T4 middle promoter. As expected, AsiA binds to and remodels σ region 4 to prevent its contact with host promoters. Unexpectedly, AsiA undergoes a large conformational change, takes over the job of σ region 4 and provides an anchor point for the upstream double-stranded DNA. Because σ region 4 is conserved among bacteria, other transcription factors may use the same strategy to alter the landscape of transcription immediately. Together, the structure provides a foundation for understanding σ appropriation and transcription activation.
Highlights
Bacterial transcription is catalyzed by RNA polymerase (RNAP), which consists of ␣I, ␣II, ,  and subunits
To obtain a structure of appropriation complex, we used a nucleic-acid scaffold corresponding to positions −45 to +15 of a T4 middle promoter (PuvsX, positions numbered relative to the transcription start site; Figure 1A)
The C-terminal half of AsiA interacts with the CTR and flap tip helix (FTH) of RNAP directly
Summary
Bacterial transcription is catalyzed by RNA polymerase (RNAP), which consists of ␣I, ␣II, ,  and subunits. The principle factor, 70 in Escherichia coli (E. coli), contacts RNAP extensively and mediates sequencespecific interactions with promoter DNA. Transcription of T4 middle genes proceeds by activation of T4 middle promoters through a process called appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA [11,14]. In this process, the co-activator AisA binds to and remodels R4, which allows the activator MotA to interact with R4, as well. The conformational change of R4 prevents its normal contact with the promoter -35 element, so the transcription of host genes is inhibited
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