Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

Abstract

Transcriptional initiation invariably involves the transitionRegulation of transcription enables cells to adapt and differentiate through coordination of protein synthesis. Two major mechanisms exist to control gene transcription as follows: one through recruitment to or preventing the RNA polymerase (RNAP) 4 binding to promoter DNA and one through activation or inhibition of the RNA polymerase activity. Despite the variations in subunit numbers that constitute the basal transcription machinery, RNA polymerases are structurally and functionally highly conserved in all kingdoms of life (1). During the initiation of transcription, RNA polymerases invariably melt the promoter DNA and have to engage the single-stranded template DNA in the structurally conserved catalytic cleft (2).The major variant bacterial 54 -RNAP is regulated by an activating mechanism with some resemblance to the operation of eukaryotic RNAP II. In both cases, passing from the closed to the open RNAP promoter complex requires activator proteins that hydrolyze nucleoside triphosphate to drive open RNAP promoter complex formation (3). The 54 -RNAP binds to specific promoter sites centered on positions Ϫ24 and Ϫ12 relative to the transcription start site and remains in a transcriptionally silent conformation. Open complexes of the 54 -RNAP promoter complexes are thermodynamically labile, and activation relies on the productive coupling of nucleoside triphosphate hydrolysis (energy coupling) from 54 activators to the 54 -RNAP promoter complex. The 54 activator-dependent initial events in open complex formation are mediated by structural changes between 54 and the promoter DNA, which can be melted from position Ϫ12 to Ϫ5 in a reaction that can occur independently from RNAP core determinants. The restructuring of promoter DNA by activated 54 has been termed 54 isomerization (4 -6). Following the initial events and requiring the presence of the RNAP core, the promoter DNA opening extends to position ϩ3 relative to the transcription start site (7).Energy coupling primarily involves intimate interactions between the activator and the 54 promoter complex. 54 activators, also termed enhancer-binding proteins (EBP), belong to the versatile AAAϩ protein (ATPases associated with various cellular activities) family of molecular machines (for review see Refs. 8 -10). Members of EBPs include the well studied NtrC, PspF, DctD, XylR, NifA, and DmpR proteins (11). PspF is the phage shock protein F that activates transcription of psp genes involved in the phage shock response in Escherichia coli (12, * This work was supported in part by the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence may be addressed. Tel.: 44-207-5945366; Fax: 44-207-5842056; E-mail: j.schumacher@imperial.ac.uk. 2 Recipient of EMBO F...

Mechanism of Homotropic Control to Coordinate Hydrolysis in a Hexameric AAA+ Ring ATPase

“…Because we speculated that K230 and N231 could be implicated in inter subunit communication, we tested previously described PspF 1-275 K230A , PspF 1-275 N231A and various PspF 1-275 variants for ATP inhibition. 23 Only PspF K230A showed reduced negative allostery in these experiments (Fig. 2a, rectangles) compared to PspF 1-275 , expressed as a percentage of their individual V max values ( Table 1).…”

“…We incubated a pre-formed σ 54 -nifH promoter probe complex with PspF 1-275 variants in the presence of ADP-AlF x reagents. 23 Figure 4a shows that PspF 1-275 , PspF 1-275 K230A and PspF 1-275 N231A can engage stably with a σ 54-promoter complex. These results demonstrate that PspF 1-275 K230A and PspF 1-275 N231A can bind ADP-AlF x and adopt the transition-state conformation required to stably bind σ 54 -promoter DNA.…”

“…Furthermore, low ATP turnover rates of PspF K230A are unlikely to entirely explain the lack of transcription activation activity, since turnover rates significantly lower than those seen for PspF K230A are sufficient for functional output in PspF 1-275 variants that are involved directly in catalysis. 23,32 To establish that the K-NH 3 + -2′-OH interaction is indeed required for allosteric control, we compared the ability of ATP and dATP to isomerise the σ 54 -promoter complex in nucleoside triphosphate titration experiments and time-course experiments (Fig. 5).…”

Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis

“…Extensive mutagenesis studies suggest that bEBPs remodel s54 protein via interactions with region I, the protein's N-terminal domain (Gonzalez et al 1998;Bordes et al 2004;Dago et al 2007;Schumacher et al 2007;Zhang et al 2009). Region I spans ;50 amino acids that are predicted to form an a helix; it possesses two short motifs that are suggested to make direct contact with the GAFTGA motifs of the ATPase (Merrick 1993;Zhang et al 2012).…”

Recent Advances in the Expression, Evolution, and Dynamics of Prokaryotic Genomes

“…J. Schumacher (London, United Kingdom) discussed transcriptional activation by AAAϩ enhancer binding proteins in an informative presentation using PspF (named for phage shock protein F) of E. coli as an example. PspF drives the transition of the sigma 54 -RNA polymerase-promoter complex from a closed conformation to a transcriptionally active open conformation in a reaction requiring ATP hydrolysis (82,83). The AAAϩ enhancer binding proteins are part of a larger family of AAAϩ proteins involved in diverse cellular activities.…”