A prehydrolysis state of an AAA+ ATPase supports transcription activation of an enhancer-dependent RNA polymerase.
ATP hydrolysis-dependent molecular machines and motors often drive regulated conformational transformations in cell signaling and gene regulation complexes. Conformational reorganization of a gene regulation complex containing the major variant form of bacterial RNA polymerase (RNAP), Esigma(54), requires engagement with its cognate ATP-hydrolyzing activator protein. Importantly, this activated RNAP is essential for a number of adaptive responses, including those required for bacterial pathogenesis. Here we characterize the initial encounter between the enhancer-dependent Esigma(54) and its cognate activator AAA+ ATPase protein, before ADP+P(i) formation, using a small primed RNA (spRNA) synthesis assay. The results show that in a prehydrolysis state, sufficient activator-dependent rearrangements in Esigma(54) have occurred to allow engagement of the RNAP active site with single-stranded promoter DNA to support spRNA synthesis, but not to melt the promoter DNA. This catalytically competent transcription intermediate has similarity with the open promoter complex, in that the RNAP dynamics required for DNA scrunching should be occurring. Significantly, this work highlights that prehydrolysis states of ATPases are functionally important in the molecular transformations they drive.
Burrows
Patricia C
PC
Division of Biology, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, United Kingdom. pcb1@imperial.ac.uk
Joly
Nicolas
N
Buck
Martin
M
D002855:Chromatography, Thin Layer; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism*; D006868:Hydrolysis; D009691:Nucleic Acid Denaturation; D051877:RNA Polymerase Sigma 54 / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology*
D015534:Trans-Activators; D051877:RNA Polymerase Sigma 54