Interactions of Upstream and Downstream Promoter Regions with RNA Polymerase are Energetically Coupled and a Target of Regulation in Transcription Initiation
التفاصيل البيبلوغرافية
العنوان:
Interactions of Upstream and Downstream Promoter Regions with RNA Polymerase are Energetically Coupled and a Target of Regulation in Transcription Initiation
During transcription initiation, the RNA polymerase holoenzyme (RNAP) and the promoter form an open complex. For many promoters, this interaction involves upstream DNA wrapping, downstream promoter bending, and associated large-scale protein rearrangements. Although these processes have been reported across the life kingdom, their structure, energetics, and role in transcription remain an area of active research. Using optical tweezers, we find that these processes become energetically and reversibly coupled after the formation of the open promoter complex, providing the main contribution to their stability. Using electron microscopy and single particle analysis, we find that the interaction encompasses from positions −76 to +18 along the template, that it involves an overall DNA bent angle of ~245°, and that the upstream wrapping is enabled by interactions between the C-terminal domains of RNAP’s alpha subunits and proximal and middle upstream promoter regions. The energy associated with upstream wrapping, downstream bending and its coupling to downstream rearrangements does not require specific upstream promoter sequence, and correlate positively with the rate of transcription DNA bubble formation as reported by a real-time fluorescence assay. Our results suggest that the coupling between upstream and downstream events are part of a cis-regulatory network established after the opening of the DNA bubble, that could furnish a control mechanism of gene expression by protein factors and regulatory metabolites.SummaryThe first step of gene expression involves transcription of DNA into RNA by RNA polymerase (RNAP). RNAP recognizes a promoter sequence forming the transcriptionally active open complex. For several promoters, DNA wraps around the RNAP. We find that upstream wrapping contacts are energetically coupled and occur cooperatively with downstream rearrangements in the open complexes, providing the largest contribution to their stability. We also determined that upstream wrapping is enabled by interactions between non-specific upstream promoter regions and RNAP α subunit C-terminal domains. Significantly, the strength of these contacts correlates with the rate of DNA bubble opening, and is regulated by factors such as the transcriptional regulator ppGpp. We suggest that any modulator altering upstream wrapping and downstream rearrangements could finely tune gene expression in response to the needs of the cell
