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bioRxiv · 10.1101/2025.02.15.638424

Subunit specialisation in AAA+ proteins and substrate unfolding during transcription complex remodelling

Gao, F.·Ye, F.·Buck, M.·Zhang, X.
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Abstract

Bacterial RNA polymerase (RNAP) is a multi-subunit enzyme that copies DNA into RNA in a process known as transcription. Bacteria use {sigma} factors to recruit RNAP to promoter regions of genes that need to be transcribed, with 60% bacteria containing at least one specialized {sigma} factor {sigma}54. {sigma}54 recruits RNAP to promoters of genes associated with stress responses and forms a stable closed complex that does not spontaneously isomerize to the open state where promoter DNA is melted out and competent for transcription. The {sigma}54-mediated open complex formation requires specific AAA+ proteins (ATPases Associated with diverse cellular Activities) known as bacterial enhancer-binding proteins (bEBPs). We have now obtained structures of new intermediate states of bEBP-bound complexes during transcription initiation, which elucidate the mechanism of DNA melting driven by ATPase activity of bEBPs and suggest a mechanistic model that couples the ATP hydrolysis cycle within the bEBP hexamer with {sigma}54 unfolding. Our data reveal that bEBP forms a non-planar hexamer with the hydrolysis-ready subunit located at the furthest/highest point of the spiral hexamer relative to the RNAP. ATP hydrolysis induces conformational changes in bEBP that drives a vectoral transiting of the regulatory N-terminus of {sigma}54 into the bEBP hexamer central pore causing the partial unfolding of {sigma}54, while forming specific bEBP contacts with promoter DNA. Furthermore, our data suggest a mechanism of AAA+ protein that is distinct from the hand-over-hand mechanism proposed for many AAA+ proteins, highlighting the versatile mechanisms utilized by the large protein family.

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Gao, F., Ye, F., Buck, M., Zhang, X.. 2025-02-16. Subunit specialisation in AAA+ proteins and substrate unfolding during transcription complex remodelling. https://doi.org/10.1101/2025.02.15.638424

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