Coupling sigma factor conformation to RNA polymerase reorganisation for DNA melting.
ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (sigma54-RNAP, Esigma54) and a slowly hydrolysed ATP analogue (ATPgammaS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of sigma54-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Esigma54 occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity sigma54 factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic beta/beta' subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Esigma54 promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the beta' jaw domain, thereby allowing acquisition of the open complex status.
Division of Biology, Department of Life Sciences, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.
Patricia C
PC
Burrows
Nicolas
N
Joly
Wendy V
WV
Cannon
Beatriz P
BP
Cámara
Mathieu
M
Rappas
Xiaodong
X
Zhang
Kathleen
K
Dawes
B Tracy
BT
Nixon
Siva R
SR
Wigneshweraraj
Martin
M
Buck
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000378:metabolism; D006868:Hydrolysis; D007711:Klebsiella pneumoniae; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D051877:RNA Polymerase Sigma 54 / Q000737:chemistry* / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D021122:Protein Subunits; C098954:PspF protein, E coli; D015534:Trans-Activators; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D051877:RNA Polymerase Sigma 54; D000251:Adenosine Triphosphatases