The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli : Identifying a surface that binds σ ⁵⁴

Abstract

Members of the protein family called ATPases associated with various cellular activities (AAA ؉ ) play a crucial role in transforming chemical energy into biological events. AAA ؉ proteins are complex molecular machines and typically form ring-shaped oligomeric complexes that are crucial for ATPase activity and mechanism of action. The Escherichia coli transcription activator phage shock protein F (PspF) is an AAA ؉ mechanochemical enzyme that functions to sense and relay the energy derived from nucleoside triphosphate hydrolysis to catalyze transcription by the 54 -RNA polymerase. Closed promoter complexes formed by the 54 -RNA polymerase are substrates for the action of PspF. By using a protein fragmentation approach, we identify here at least one 54 -binding surface in the PspF AAA ؉ domain. Results suggest that ATP hydrolysis by PspF is coupled to the exposure of at least one 54 -binding surface. This nucleotide hydrolysis-dependent presentation of a substrate binding surface can explain why complexes that form between 54 and PspF are transient and could be part of a mechanism used generally by other AAA ؉ proteins to regulate activity.

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

“…Mutagenesis and Protein Purifications-Plasmid pPB1 (14) encoding E. coli PspF residues 1-275 (PspF with an N-terminal His 6 tag in pET28b ϩ ) was mutagenized (QuikChange mutagenesis kit, Stratagene) to obtain the desired single amino acid substitutions in pspF, resulting in pPB1(T148A), pPB1(N149A), and pPB1(N149S). All PspF 1-275 proteins were purified in the same manner and as described previously (24).…”

“…The AAAϩ domain (see Fig. 1 for AAAϩ domain features) of the 54 activator PspF (PspF ) is necessary and sufficient to activate transcription of the 54 -RNAP in vitro and in vivo (14,15). The 54 activator signature sequence GAFTGA loop 1 directly binds to the region I of 54 , which is involved in maintaining a closed 54 -RNAP promoter complex (16,17 (19,20).…”

Investigating protein–protein interfaces in bacterial transcription complexes: a fragmentation approach

“…Bordes et al, (1) proposed that, on the binding and subsequent hydrolysis of NTPs, PspF is able to adopt a different conformation, and hence a new functional state, so as to interact with its target substrate, s 54 within a closed promoter complex. Depending on the state of the nucleotide bound, the sequences that are proposed to interact with s 54 are either masked or presented.…”

“…(22) Therefore productive interactions between the EBP and s 54 are conditioned by the state of the nucleotide bound to the activator, seemingly by the sensing of the g-phosphate of ATP. (9) The recent study by Bordes et al (1) determined that a unique sequence present within s 54 -dependent EBPs is involved in directly binding s 54 and that this sequence is not static, but subject to movement in a nucleotide-dependent fashion. It was also proposed that a similar mechanism might be utilised by other molecular machines belonging to the AAAþ superfamily.…”

“…s 54 within closed complexes) binding interface lies. In order to better understand the intimate interactions made between the EBP and s 54 -RNAP, Bordes and colleagues (1) conducted protein fragmentation studies with the E. coli EBP phage shock protein F (PspF), a transcription regulator involved in stress response. (21,22) The fragmentation approach was adopted to overcome the problem of overlapping functionality associated with full-length protein-the activities of the full-length protein are interlinked.…”