Comparative analysis of activator-Esigma54 complexes formed with nucleotide-metal fluoride analogues.
Bacterial RNA polymerase (RNAP) containing the major variant sigma(54) factor forms open promoter complexes in a reaction in which specialized activator proteins hydrolyse ATP. Here we probe binding interactions between sigma(54)-RNAP (Esigma(54)) and the ATPases associated with various cellular activities (AAA+) domain of the Escherichia coli activator protein, PspF, using nucleotide-metal fluoride (BeF and AlF) analogues representing ground and transition states of ATP, which allow complexes (that are otherwise too transient with ATP) to be captured. We show that the organization and functionality of the ADP-BeF- and ADP-AlF-dependent complexes greatly overlap. Our data support an activation pathway in which the initial ATP-dependent binding of the activator to the Esigma(54) closed complex results in the re-organization of Esigma(54) with respect to the transcription start-site. However, the nucleotide-dependent binding interactions between the activator and the Esigma(54) closed complex are in themselves insufficient for forming open promoter complexes when linear double-stranded DNA is present in the initial closed complex.
Burrows
Patricia C
PC
Department of Life Sciences, Division of Biology, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.
Joly
Nicolas
N
Nixon
B Tracy
BT
Buck
Martin
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D017607:Aluminum Compounds / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005459:Fluorides / Q000737:chemistry; D008958:Models, Molecular; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54 / Q000737:chemistry* / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic
D017607:Aluminum Compounds; D029968:Escherichia coli Proteins; C098954:PspF protein, E coli; D015534:Trans-Activators; D000255:Adenosine Triphosphate; D004247:DNA; D051877:RNA Polymerase Sigma 54; D005459:Fluorides; C032311:aluminum fluoride