DNA melting within a binary sigma(54)-promoter DNA complex.
The final sigma(54) subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription. Here we show that final sigma(54) is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found in closed complexes. Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream from the nucleation point toward the transcription start site. We show that final sigma(54) contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require Region I. It seems that final sigma(54) binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change in final sigma(54) to reveal its DNA-melting activity. Results with the holoenzyme bound to early melted DNA suggest an ordered series of events in which changes in core to final sigma(54) interactions and final sigma(54)-DNA interactions occur in response to activator to allow final sigma(54) isomerization and the holoenzyme to progress from the closed complex to the open complex.
Cannon
W
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Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom.
Gallegos
M T
MT
Buck
M
M
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D020035:Holoenzymes / Q000378:metabolism; D007536:Isomerism; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010088:Oxidoreductases / Q000235:genetics; D011196:Potassium Permanganate / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000235:genetics; D013816:Thermodynamics; D015534:Trans-Activators / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D020035:Holoenzymes; C098954:PspF protein, E coli; D012808:Sigma Factor; D015534:Trans-Activators; C497751:rpoN protein, E coli; D011196:Potassium Permanganate; D004247:DNA; D010088:Oxidoreductases; C029130:nitrogenase reductase; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D003850:Deoxyribonuclease I