Purification and Properties of the NAD+: Protein ADP‐ribosyltransferase Responsible for the T4‐Phage‐Induced Modification of the œ Subunit of DNA‐Dependent RNA Polymerase of Escherichia coli

Abstract

The NAD+: protein ADP‐ribosyltransferase responsible for the bacteriophage‐T4‐induced modification of RNA polymerase has been purified 100–200‐fold from Escherichia coli B/r infected with the phage T4. The enzyme has a molecular weight around 26000. It does not require magnesium ions and is protected by sulfhydryl reagents. The temperature optimum is around 20°C. The reaction is inhibited at increased ionic strength and by the reaction products nicotinamide and T4‐modified RNA polymerase. The reaction is irreversible. The only reaction products formed to a significant extent in a crude extract are T4‐modified a subunit and one smaller ADP‐ribosylated peptide. As in vivo, the reaction is complete. Both œ subunits in the RNA polymerase monomer are ADP‐ribosylated in one and the same position. The product of modification in vitro appears to be identical with that of modification in vivo. ADP‐ribose has been obtained by hydrolysis both of T4‐modified œ subunit and of the reaction product of egg‐white lysozyme, NAD+ and the transferase responsible for bacteriophage‐T4‐induced alteration of host proteins [Rohrer, H., Zillig, W., and Mailhammer, R. (1975) Eur. J. Biochem. 60, 227–238]. The amino acid carrying the ADP‐ribosyl residue appears to be arginine. T4‐modified œ subunit and the major species of T4‐altered œ subunit are identical. An NAD+: protein ADP‐ribosyltransferase using E. coli proteins and egg‐white lysozyme but no RNA polymerase subunit as acceptor(s) has been purified from uninfected E. coli cells. Copyright © 1977, Wiley Blackwell. All rights reserved