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-Structure paper
Title | Subunit specialization in AAA+ proteins and substrate unfolding during transcription complex remodeling. |
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Journal, issue, pages | Proc Natl Acad Sci U S A, Vol. 122, Issue 17, Page e2425868122, Year 2025 |
Publish date | Apr 29, 2025 |
![]() | Forson Gao / Fuzhou Ye / Martin Buck / Xiaodong Zhang / ![]() |
PubMed Abstract | Bacterial RNA polymerase (RNAP) is a multisubunit enzyme that copies DNA into RNA in a process known as transcription. Bacteria use σ factors to recruit RNAP to promoter regions of genes that need ...Bacterial RNA polymerase (RNAP) is a multisubunit enzyme that copies DNA into RNA in a process known as transcription. Bacteria use σ factors to recruit RNAP to promoter regions of genes that need to be transcribed, with 60% bacteria containing at least one specialized σ factor, σ. σ 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 σ-mediated open complex formation requires specific AAA+ proteins (TPases ssociated with diverse cellular ctivities) 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 σ 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 σ into the bEBP hexamer central pore causing the partial unfolding of σ, 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. |
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Methods | EM (single particle) |
Resolution | 3.9 - 9.4 Å |
Structure data | EMDB-52914, PDB-9q91: EMDB-52916, PDB-9q93: EMDB-52917, PDB-9q94: EMDB-52918, PDB-9q95: EMDB-52919, PDB-9q96: ![]() EMDB-52922: PspF(1-275) subtracted focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -10 to -1 ![]() EMDB-52923: PspF(1-275)-DNA-sigma54(R1) focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -10 to -1 - open clamp conformation ![]() EMDB-52924: Consensus map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -10 to -1 - Open clamp conformation ![]() EMDB-52925: PspF(1-275)-DNA-sigma54(R1) focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -10 to -1 - Closed clamp conformation ![]() EMDB-52926: RNA polymerase-sigma54-DNA focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -10 to -1 - Closed clamp conformation ![]() EMDB-52927: Consensus map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -10 to -1 - Closed clamp conformation ![]() EMDB-52928: RNA polymerase-sigma54-DNA subtracted focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - open clamp ![]() EMDB-52929: RNA polymerase-sigma54-DNA subtracted focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - closed clamp ![]() EMDB-52930: PspF(1-275) subtracted focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 ![]() EMDB-52931: PspF(1-275)-DNA-sigma54(R1) focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 6 ![]() EMDB-52932: RNA polymerase-sigma54-DNA focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 6 ![]() EMDB-52933: Consensus map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 6 ![]() EMDB-52934: PspF(1-275)-DNA-sigma54(R1) focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 5 ![]() EMDB-52935: RNA polymerase-sigma54-DNA focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 5 ![]() EMDB-52936: Consensus map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 5 ![]() EMDB-52937: PspF(1-275)-DNA-sigma54(R1) focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 1 ![]() EMDB-52938: PspF(1-275)-DNA-sigma54(R1) focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 4 ![]() EMDB-52939: RNA polymerase-sigma54-DNA focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 4 ![]() EMDB-52940: Consensus map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 4 ![]() EMDB-52941: Consensus map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 3 ![]() EMDB-52942: RNA polymerase-sigma54-DNA focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 3 ![]() EMDB-52943: PspF(1-275)-DNA-sigma54(R1) focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 3 ![]() EMDB-52944: RNA polymerase-sigma54(R1)-DNA focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 2 ![]() EMDB-52945: Consensus map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 2 ![]() EMDB-52946: PspF(1-275)-DNA-sigma54(R1) focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 2 EMDB-52947, PDB-9q97: ![]() EMDB-52948: RNA polymerase-sigma54-DNA focussed map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 1 ![]() EMDB-52949: Consensus map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 1 EMDB-52950: Composite map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 1 |
Chemicals | ![]() ChemComp-ADP: ![]() ChemComp-AF3: ![]() ChemComp-MG: |
Source |
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![]() | TRANSCRIPTION / sigma factor / complex / AAA+ / DNA-binding protein / TRANSCRIPTION INITIATION / DNA OPENING / TRANSCRIPTION BUBBLE |