Characterization of a photoaffinity analog of UTP, 5-azido-UTP for analysis of the substrate binding site on E. coli RNA polymerase.
The substrate binding site on E. coli RNA polymerase was investigated by photoaffinity labeling with a photoaffinity analog of UTP, 5-azido-UTP. We have established that 5-azido-UTP is a substrate for RNA polymerase by specific transcription on 229 bp DNA containing the gene II promoter of M13 phage. Analysis of the initial rate of RNA synthesis gives Km(5-azido-UTP) approximately 80 microM. Photolabeling with varying concentrations of 5-azido-UTP follows a saturation curve with the midpoint occurring at a 5-azido-UTP concentration of 65 microM near to the Km obtained by kinetic analysis. 5-Azido-UTP photolabels the beta', beta, and sigma subunits to about the same extent, both in the presence (33, 31, and 36%) and absence (35, 30 and 35%) of DNA. This labeling pattern is somewhat different from that obtained with 8-azido-ATP (beta' greater than sigma much greater than beta greater than alpha).
D000345:Affinity Labels; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D010777:Photochemistry; D010957:Plasmids; D012313:RNA / Q000096:biosynthesis; D014158:Transcription, Genetic
D000345:Affinity Labels; D001386:Azides; D003856:Deoxyuracil Nucleotides; C048865:5-azido-2'-deoxyuridine 5'-triphosphate; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases