Characterization of the closed complex intermediate formed during transcription initiation by Escherichia coli RNA polymerase.
We have carried out detailed DNase I footprinting studies of the closed complex formed on the phage lambda prmup-1 Delta265 promoter under reaction conditions such that the contribution of the open complex to the footprint was negligible. Detailed quantification shows that the closed complex detected has the same binding constant as that determined in kinetic studies. The footprinting pattern of the closed complex shows major differences from that of the open complex. Not only is it about 20 base pairs shorter, there are also many fewer positions being protected around and upstream of the -35 region. We have derived potential contact regions in the closed and open complexes based on the DNase I footprinting patterns, and confirmed the contact region for the open complex by hydroxyl radical footprinting. One important finding is that most of the essential contacts with the phosphate groups in the -35 region are formed during the isomerization step, a conclusion consistent with our kinetic data showing that this step is salt dependent on this promoter. In addition, we found that the derived contact regions for the closed and open complexes are offset by about three base pairs in the -35 region, which suggests a shift of the contact during isomerization. Finally, we found that the footprinting pattern of the complex formed at 4 degreesC has some similarities to as well as differences from the closed complex formed under standard transcription conditions.
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
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D001426:Bacterial Proteins / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004279:DNA, Viral / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics*; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004279:DNA, Viral; D017665:Hydroxyl Radical; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I