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TitleSubunit specialization in AAA+ proteins and substrate unfolding during transcription complex remodeling.
Journal, issue, pagesProc Natl Acad Sci U S A, Vol. 122, Issue 17, Page e2425868122, Year 2025
Publish dateApr 29, 2025
AuthorsForson Gao / Fuzhou Ye / Martin Buck / Xiaodong Zhang /
PubMed AbstractBacterial 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.
External linksProc Natl Acad Sci U S A / PubMed:40273105
MethodsEM (single particle)
Resolution3.9 - 9.4 Å
Structure data

EMDB-52914, PDB-9q91:
CryoEM structure of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 6
Method: EM (single particle) / Resolution: 7.2 Å

EMDB-52916, PDB-9q93:
CryoEM structure of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 4
Method: EM (single particle) / Resolution: 6.6 Å

EMDB-52917, PDB-9q94:
CryoEM structure of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 3
Method: EM (single particle) / Resolution: 5.8 Å

EMDB-52918, PDB-9q95:
CryoEM structure of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -10 to -1
Method: EM (single particle) / Resolution: 6.8 Å

EMDB-52919, PDB-9q96:
Cryo-EM Structure of Bacterial RNA polymerase-sigma54 transcription open complex with wild type sigma54, from RPi(-10-1)
Method: EM (single particle) / Resolution: 4.6 Å

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
Method: EM (single particle) / Resolution: 4.7 Å

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
Method: EM (single particle) / Resolution: 9.4 Å

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
Method: EM (single particle) / Resolution: 5.3 Å

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
Method: EM (single particle) / Resolution: 6.2 Å

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
Method: EM (single particle) / Resolution: 5.6 Å

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
Method: EM (single particle) / Resolution: 6.8 Å

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
Method: EM (single particle) / Resolution: 4.2 Å

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
Method: EM (single particle) / Resolution: 3.9 Å

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
Method: EM (single particle) / Resolution: 4.7 Å

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
Method: EM (single particle) / Resolution: 7.07 Å

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
Method: EM (single particle) / Resolution: 6.8 Å

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
Method: EM (single particle) / Resolution: 7.2 Å

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
Method: EM (single particle) / Resolution: 6.1 Å

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
Method: EM (single particle) / Resolution: 5.9 Å

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
Method: EM (single particle) / Resolution: 6.8 Å

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
Method: EM (single particle) / Resolution: 7.6 Å

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
Method: EM (single particle) / Resolution: 7.1 Å

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
Method: EM (single particle) / Resolution: 6.3 Å

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
Method: EM (single particle) / Resolution: 6.6 Å

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
Method: EM (single particle) / Resolution: 5.8 Å

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
Method: EM (single particle) / Resolution: 5.3 Å

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
Method: EM (single particle) / Resolution: 5.7 Å

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
Method: EM (single particle) / Resolution: 4.5 Å

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
Method: EM (single particle) / Resolution: 4.6 Å

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
Method: EM (single particle) / Resolution: 6.6 Å

EMDB-52947, PDB-9q97:
CryoEM structure of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 2
Method: EM (single particle) / Resolution: 4.6 Å

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
Method: EM (single particle) / Resolution: 6.8 Å

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
Method: EM (single particle) / Resolution: 6.8 Å

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
PDB-9q98: CryoEM structure of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 1
Method: EM (single particle) / Resolution: 8.3 Å

Chemicals

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

ChemComp-AF3:
ALUMINUM FLUORIDE

ChemComp-MG:
Unknown entry

Source
  • escherichia coli k-12 (bacteria)
  • klebsiella pneumoniae (bacteria)
KeywordsTRANSCRIPTION / sigma factor / complex / AAA+ / DNA-binding protein / TRANSCRIPTION INITIATION / DNA OPENING / TRANSCRIPTION BUBBLE

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