Coupling nucleotide hydrolysis to transcription activation performance in a bacterial enhancer binding protein.
The bacterial enhancer binding proteins (bEBP) are members of the AAA+ protein family and have a highly conserved 'DE' Walker B motif thought to be involved in the catalytic function of the protein with an active role in nucleotide hydrolysis. Based on detailed structural data, we analysed the functionality of the conserved 'DE' Walker B motif of a bEBP model, phage shock protein F (PspF), to investigate the role of these residues in the sigma(54)-dependent transcription activation process. We established their role in the regulation of PspF self-association and in the relay of the ATPase activity to the remodelling of an RNA polymerase.promoter complex (Esigma(54).DNA). Specific substitutions of the conserved glutamate (E) allowed the identification of new functional ATP.bEBP.Esigma(54) complexes which are stable and transcriptionally competent, providing a new tool to study the initial events of the sigma(54)-dependent transcription activation process. In addition, we show the importance of this glutamate residue in sigma(54).DNA conformation sensing, permitting the identification of new intermediate stages within the transcription activation pathway.
Joly
Nicolas
N
Division of Biology, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.
Rappas
Mathieu
M
Wigneshweraraj
Siva R
SR
Zhang
Xiaodong
X
Buck
Martin
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051877:RNA Polymerase Sigma 54 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009711:Nucleotides; C098954:PspF protein, E coli; D015534:Trans-Activators; D000255:Adenosine Triphosphate; D051877:RNA Polymerase Sigma 54