title: Sensor I threonine of the AAA+ ATPase transcriptional activator PspF is involved in coupling nucleotide triphosphate hydrolysis to the restructuring of sigma 54-RNA polymerase.
abstract: Transcriptional initiation invariably involves the transition from a closed RNA polymerase (RNAP) promoter complex to a transcriptional competent open complex. Activators of the bacterial sigma(54)-RNAP are AAA+ proteins that couple ATP hydrolysis to restructure the sigma(54)-RNAP promoter complex. Structures of the sigma(54) activator PspF AAA+ domain (PspF(1-275)) bound to sigma(54) show two loop structures proximal to sigma(54) as follows: the sigma(54) contacting the GAFTGA loop 1 structure and loop 2 that classifies sigma(54) activators as pre-sensor 1 beta-hairpin AAA+ proteins. We report activities for PspF(1-275) mutated in the AAA+ conserved sensor I threonine/asparagine motif (PspF(1-275)(T148A), PspF(1-275)(N149A), and PspF(1-275)(N149S)) within the second region of homology. We show that sensor I asparagine plays a direct role in ATP hydrolysis. However, low hydrolysis rates are sufficient for functional output in vitro. In contrast, PspF(1-275)(T148A) has severe defects at the distinct step of sigma(54) promoter restructuring. This defect is not because of the failure of PspF(1-275)(T148A) to stably engage with the closed sigma(54) promoter, indicating (i) an important role in ATP hydrolysis-associated motions during energy coupling for remodeling and (ii) distinguishing PspF(1-275)(T148A) from PspF(1-275) variants involved in signaling to the GAFTGA loop 1, which fail to stably engage with the promoter. Activities of loop 2 PspF(1-275) variants are similar to those of PspF(1-275)(T148A) suggesting a functional signaling link between Thr(148) and loop 2. In PspF(1-275) this link relies on the conserved nucleotide state-dependent interaction between the Walker B residue Glu(108) and Thr(148). We propose that hydrolysis is relayed via Thr(148) to loop 2 creating motions that provide mechanical force to the GAFTGA loop 1 that contacts sigma(54).
authors.affiliation: Division of Biology, Imperial College London, London SW7 2AZ, United Kingdom. Electronic address: j.schumacher@imperial.ac.uk.
authors.forename: Jörg
authors.initials: J
authors.lastname: Schumacher
authors.affiliation: Division of Biology, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Joly
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Rappas
authors.affiliation: Division of Biology, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Dominic
authors.initials: D
authors.lastname: Bradley
authors.affiliation: Division of Biology, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Siva R
authors.initials: SR
authors.lastname: Wigneshweraraj
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Division of Biology, Imperial College London, London SW7 2AZ, United Kingdom. Electronic address: m.buck@imperial.ac.uk.
authors.forename: Martin
authors.initials: M
authors.lastname: Buck
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D006868:Hydrolysis; D051877:RNA Polymerase Sigma 54 / Q000737:chemistry / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000502:physiology*; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*
chemical_list: D029968:Escherichia coli Proteins; C098954:PspF protein, E coli; D015534:Trans-Activators; C497751:rpoN protein, E coli; D013912:Threonine; D000255:Adenosine Triphosphate; D051877:RNA Polymerase Sigma 54