Affinity labeling of a cysteine at or near the catalytic center of Escherichia coli B DNA-dependent RNA polymerase.

ABSTRACT

9-beta-D-Arabinofuranosyl-6-thiopurine was used to affinity label DNA-dependent RNA polymerase isolated from Escherichia coli B. This substrate analogue displayed competitive type inhibition which could be reversed by addition of a thiol reagent, such as dithiothreitol, while exposure to hydrogen peroxide, a mild oxidizing agent, caused an increase in both the inhibitory and enzyme binding capability of arabinofuranosyl thiopurine. Chromatographic analysis of the products obtained by pronase digestion of the 9-beta-D-arabinofuranosyl-6-[35S]thiopurine-enzyme complex suggests that disulfide bond formation occurs between the inhibitor and a cysteine residue located in or near the active center of the enzyme. In addition, polyacrylamide gel electrophoresis indicated that the arabinofuranosyl thiopurine moeity was bound to the beta' subunit of the enzyme.