Conformational changes of E. coli RNA polymerase during transcription initiation.
Escherichia coli RNA polymerase-promoter complex undergoes a multistep process to initiate transcription. We have employed fluorescence spectroscopic approaches to detect the conformational states of the enzyme during this multistep process. A fluorescence assay based on the measurement of fluorescence of free and promoter-bound enzyme as a function of temperature within the range of 4 to 37 degrees C showed that, starting with initial 'closed complex', there are conformationally two distinct intermediate states of the polymerase till it attains the final form required for transcription initiation. The equilibrium from closed complex (RPc) to open complex (RPo) consists of at least the following two intermediate complexes: [formula: see text] Higher order structure of RNAP in each of these complexes was probed by means of measurement of accessibilities of the tryptophan fluorophores to the acrylamide. In the next part of the study, TbGTP, a fluorescent substrate, has been used to probe the state of active site in the enzyme for the complexes RPc, RPi1, RPi2 and RPo, respectively. From the comparison of changes in the parameters such as, fluorescence polarization anisotropy of TbGTP and its accessibility to the neutral quencher, acrylamide, in free and promoter-bound enzyme, we have further substantiated the first part of our results. Together these results suggest that formations of RPc and RPi1 do not involve radical conformational changes in the enzyme, while the enzyme undergoes major change in conformation in the steps RPil-->RPi2 and RPi2-->RPo. The strong tryptophan promoter cloned in plasmid pDR720 was chosen as a model promoter in these studies.
Sen
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Biophysics Division, Saha Institute of Nuclear Physics, Calcutta, India.
Dasgupta
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D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005454:Fluorescence Polarization; D006160:Guanosine Triphosphate / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013725:Terbium / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*
D009942:Organometallic Compounds; D013725:Terbium; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases