Subunit specialization in AAA plus proteins and substrate unfolding during transcription complex remodeling

• 成果类型: Article
• 署名作者: Gao, Forson; Ye, Fuzhou; Buck, Martin; Zhang, Xiaodong
• 署名单位: Imperial College London; Imperial College London; Francis Crick Institute; MRC Laboratory Molecular Biology; University of London; Institute of Cancer Research - UK; Royal Marsden NHS Foundation Trust
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-11263
• DOI: 10.1073/pnas.2425868122
• 发表日期: 2025-04-29
• 关键词: rna; mechanisms; binding; reveal

摘要

Bacterial RNA polymerase (RNAP) is a multisubunit enzyme that copies DNA into RNA in a process known as transcription. Bacteria use 6 factors to recruit RNAP to promoter regions of genes that need to be transcribed, with 60% bacteria containing at least one specialized 6 factor, 654. 654 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 654-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 Adenosine triphosphate (ATP) hydrolysis cycle within the bEBP hexamer with 654 unfolding. Our data reveal that bEBP forms a nonplanar 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 654 into the bEBP hexamer central pore causing the partial unfolding of 654, while forming specific bEBP contacts with promoter DNA. Furthermore, our data suggest a mechanism of the bEBP AAA+ protein that is distinct from the hand-over-hand mechanism proposed for many other AAA+ proteins, highlighting the versatile mechanisms utilized by the large protein family.