Structural studies of sigma54-mediated transcription initiation

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Transcription is an essential step in gene expression in all organisms and involves the conversion of sequential information from genomic DNA to single-stranded RNA by the enzyme RNA polymerase. Transcription initiation is the most regulated step in transcription and is essential in ensuring an organism can adapt and respond to changes in its environment and maintain homeostasis. In bacteria, transcription initiation is mediated by o factors, which are classified as housekeeping o70 family or major variant o54. o factors recruit RNA polymerase to upstream promoter regions of genes and regulate the ability for RNA polymerase to melt and load DNA into its active site to form a competent open complex.o54 promoters are associated with nutrient depletion and stress response, and o54 is unique as it recruits RNA polymerase to form a stable closed complex. o54 initially inhibits open complex formation by blocking DNA loading, as well as occupying the active site and sitting atop of the RNA exit channel. Open complex formation therefore requires ATP hydrolysis by a specialised family of clade 6 AAA+ (ATPase associated with diverse cellular activity) proteins called bacterial enhancer binding proteins. Furthermore, o54 must undergo a set of conformational changes to enable promoter escape and a transition to processive elongation.Using biochemical approaches coupled with cryo-electron microscopy, we captured multiple AAA+-engaged intermediate states to elucidate the mechanism of early DNA melting during open complex formation. Furthermore, we have determined high-resolution structures capturing the initial stages of transcription, gaining insights into how o54 is displaced during promoter escape.The work presented here deepens our understanding of o54 mediated transcription initiation, demonstrates conserved mechanisms of promoter escape in all bacteria and demonstrates a unique mechanism of transcription activation by a specialised family of AAA+ protein.