ATP-dependent transcriptional activation by bacterial PspF AAA+protein

J Mol Biol. 2004 May 14;338(5):863-75. doi: 10.1016/j.jmb.2004.02.071.

Abstract

Transcription activation by bacterial sigma(54)-dependent enhancer-binding proteins (EBPs) requires their tri-nucleotide hydrolysis to restructure the sigma(54) RNA polymerase (RNAP). EBPs share sequence similarity with guanine nucleotide binding-proteins and ATPases associated with various cellular activities (AAA) proteins, especially in the mononucleotide binding P-loop fold. Using the phage shock protein F (PspF) EBP, we identify P-loop residues responsible for nucleotide binding and hydrolysis, consistent with their roles in other P-loop NTPases. We show the refined low-resolution structure of an EBP, PspF, revealing a hexameric ring organisation characteristic of AAA proteins. Functioning of EBPs involves ATP binding, higher oligomer formation and ATP hydrolysis coupled to the restructuring of the RNAP. This is thought to be a highly coordinated multi-step process, but the nucleotide-driven mechanism of oligomerisation and ATP hydrolysis is little understood. Our kinetic and structural data strongly suggest that three PspF dimers assemble to form a hexamer upon nucleotide binding. During the ATP hydrolysis cycle, both ATP and ADP are bound to oligomeric PspF, in line with a sequential hydrolysis cycle. We identify a putative R-finger, and show its involvement in ATP hydrolysis. Substitution of this arginine residue results in nucleotide-independent formation of hexameric rings, structurally linking the putative R-finger and, by inference, a specific nucleotide interaction to the control of PspF oligomerisation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / analogs & derivatives
  • Adenosine Triphosphate / metabolism*
  • Bacterial Proton-Translocating ATPases / metabolism*
  • DNA-Binding Proteins*
  • DNA-Directed RNA Polymerases / metabolism
  • Escherichia coli Proteins / metabolism*
  • Models, Molecular
  • Nucleotides / metabolism
  • Organometallic Compounds / metabolism
  • RNA Polymerase Sigma 54
  • Sigma Factor / metabolism
  • Trans-Activators / metabolism*
  • Transcription, Genetic*

Substances

  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Nucleotides
  • Organometallic Compounds
  • PspF protein, E coli
  • Sigma Factor
  • Trans-Activators
  • rpoN protein, E coli
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • DNA-Directed RNA Polymerases
  • RNA Polymerase Sigma 54
  • Bacterial Proton-Translocating ATPases