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

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 to be transcribed, with 60% bacteria containing at least one specialized σ factor, σ⁵⁴. σ⁵⁴ 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 σ⁵⁴-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 σ⁵⁴ 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 σ⁵⁴ into the bEBP hexamer central pore causing the partial unfolding of σ⁵⁴, 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.

Keywords: AAA+ ATPases; DNA opening; RNA polymerase; protein unfolding and remodeling; transcription initiation.

Fig. 1.

Structural analysis of bEBP hexamer in the transcription intermediate state of RNAP-σ⁵⁴-PspF_1-275 with DNA mismatched at −12/−11. (A) overview of the hexamer and its arrangement with DNA and σ⁵⁴ RI. (B) functional motifs and secondary structures of bEBP AAA+ domains. (C) nucleotide binding pocket, with key residues in nucleotide binding and hydrolysis labeled. (D–I) density corresponding to nucleotides in each protomer with key catalytic residues labeled.

Fig. 2.

Protomer conformations and their functional state. (A) L1 loops of protomers track σ⁵⁴ N terminus in the hexamer pore with protomer 1 at the higher position in the helical spiral. (B) conformations and interactions of L1 (brown) and L2 (blue) loops between protomer 1 and protomer 2. (C) Overlays between protomer 1 (ATP*) and protomer 6 (ADP) reveal the conformational changes upon ATP hydrolysis. (D) overlay of protomer 1 and protomer 2 shows the overall similarities in their conformation and different conformations of the L1 loops.

Fig. 3.

Proposed ATP hydrolysis cycle and conformational changes within PspF_1-275 hexamer. (A) protomers 1, 2, and 6 in the hexamer and conformational changes in protomer 1 upon hydrolysis. Insets: left–ATP hydrolysis of protomer 1 would cause it to adopt conformation of protomer 6 (arrow), causing a steric clash with protomer 2; right -upon hydrolysis, protomer 1 could then move (arrows) toward protomer 6. (B) proposed ATP hydrolysis and nucleotide-state changes within the hexamer. (C) ATP hydrolysis of protomer 1 would pull σ⁵⁴ N terminus, (D) proposed positional changes in the hexamer spiral upon ATP hydrolysis of protomer 1.

Fig. 4.

Structures of RPi (−11/−8) and RPi (−10/−1). (A) DNA substrates used to mimic partially or fully melted transcription bubble. (B) comparisons of the PspF_1-275 hexamer in relation to RNAP in the RPi complexes. (C) comparisons of DNA trajectories in these complexes. (D) additional interactions between PspF_1-275 and σ⁵⁴ RpoN domain are observed in RPi(−11/−8) and RPi(−10/−1).

Fig. 5.

ATP hydrolysis couples to σ⁵⁴ N terminus translocation and RI-H1 unfolding. (A) PspF_1-275 hexamer is arranged differently in these complexes, showing as the distances between equivalent elements in protomer 1 and protomer 6. Density for RI-H1 is also progressively worse from RPi(−12/−11) to RPi(−11/−8) and RPi(−10/−1), (B) cartoon depictions of the protomers in the hexamer spiral. (C) Top views of the hexamer showing the relative positions of the protomers in the spiral. Dashed lines indicate boundaries between protomers 1 and 6. (D) density for σ⁵⁴ N terminus, indicating that N terminus has been translocated further into the hexamer from RPi(−12/−11) to RPi(−11/−8) and RPi(−12/−11).