[Modification of the RNA-polymerase of Escherichia coli by diethylpyrocarbonate].
E. coli DNA dependent RNA polymerase was modified by diethylpyrocarbonate. Optical and kinetic properties of the reaction were studied. More than 90% of RNA polymerase activity is inhibited by introduction of 9--11 ethoxyformyl groups per enzyme molecule without loss of its ability to bind DNA template. Furthermore the modified enzyme is able to form tight complexes with DNA and to compete with native enzyme for the formation of rifampicin-resistant complex. The ratio of the complex formation constants for the native and modified enzyme was determined to be equal to 10. The enzyme modified to such extent loses the activity in DNA dependent RNA as well as pppApU synthesis. Vmax value rather than Km value for both ATP and UTP decreases following the modification reaction. Incubation of the enzyme modified to the 10% of residual activity with 0.2 M hydroxylamine for 2 hours results in restoration of RNA polymerase activity. Most but not all of the modified histidyl residues restore their native structure. Two of 13 histidyl residues were modified irreversibly due to Bamberger's cleavage reaction, but these two residues were found to be unessential for RNA polymerase activity. Reaction with higher concentration of the diethylpyrocarbonate induces modification of more than 15--20 histidyl residues and leads to irreversible inactivation of the enzyme. Nevertheless the modification of the additional histidyl redidues was reversible as well as the modification of the first 11 residues. RNA polymerase modified to such extent loses the ability to bind DNA. Preformation of the initiated ternary complex of RNA polymerase with template and product fails to protect the enzyme from reversible inactivation at a low reagent concentration, but markedly decreases the rate of the irreversible and unspecific modification of sulfhydryl or amino groups of the enzyme. Reaction with the ternary complex results in reversible inactivation of the enzyme with respect to elongation of RNA chains as well as the pyrophosphate exchange reaction. The complex itself was, however, completely stable under the reaction conditions and the enzyme subunit structure was also conserved after the reaction. Evidently, the mild modification of the histidyl residues with diethylpyrocarbonate selectively inhibits RNA chain elongation.
D055598:Chemical Phenomena; D002621:Chemistry; D004247:DNA; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors; D004047:Diethyl Pyrocarbonate; D004926:Escherichia coli / Q000201:enzymology*; D005561:Formates; D006898:Hydroxylamines
D005561:Formates; D006898:Hydroxylamines; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004047:Diethyl Pyrocarbonate
Modifikatsiia RNK-polimerazy Escherichia coli diétilpirokarbonatom.