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 ...
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).



Nucleotide Analogs / Template Lesions, Nucleotide Incorporation


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