Covalent Attachment of Ribonucleotides at 3′-Hydroxyl Ends of Deoxyribonucleic Acid Catalyzed by Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase of Escherichia coli

Abstract

Abstract DNA-dependent RNA polymerase of Escherichia coli catalyzes covalent attachment of ribonucleotides at 3'-hydroxyl termini of synthetic as well as naturally occurring DNAs. Incorporation of a single ribonucleotide occurs at 3'-hydroxyl termini of poly[d(A-T)] in the presence of ATP or UTP, at termini of poly[(dG)·(dC)] in the presence of CTP or GTP and at termini of nicked colicin E1 DNA (termini introduced by the action of pancreatic DNase) in the presence of each of the four ribonucleotides. This end addition reaction occurs optimally over a broad pH range (7 to 10) and differs from general transcription in a number of properties. It is more than 20 times less sensitive to the action of streptolydigin than RNA synthesis. Furthermore, it is inhibited only 80 % by 60 µg per ml of rifampicin while RNA synthesis is inhibited more than 95 % by 5 µ per ml of this drug. Ribonucleotide(s) can thus be attached at 3'-hydroxyl termini of DNA under conditions where synthesis of free RNA is inhibited. While the initiation reaction leading to RNA synthesis is specific for purines (ATP and GTP), the end addition reaction shows no preferences for purines. The salt sensitivity and the rate of incorporation of AMP on poly[d(A-T)] parallels that of CMP on poly[(dG)·(dC)] but not GMP. In the absence of the σ subunit of RNA polymerase AMP and UMP end addition on poly [d(A-T)] show identical kinetics. In the presence of the σ subunit, however, AMP incorporation ceases within 10 min and no further incorporation occurs for at least 30 min. On a molar basis, the amount of AMP incorporated is equal to approximately 30 to 50 % of the added RNA polymerase which, as previously shown, corresponds to the proportion of active enzyme molecules (Wickner, S., Hurwitz, J., Nath, K., and Yarbrough, L. (1972) Biochem. Biophys. Res. Commun. 48, 619–627). Estimation of active RNA polymerase molecules can thus be obtained by end addition measurement. RNA chains are formed at 3'-hydroxyl termini of poly[d(A-T)] in the presence of ATP and UTP, and at the termini of nicked colicin E1 DNA in the presence of all four ribonucleotides. This covalently attached RNA, when isolated after heating in formaldehyde, Cs2SO4 banding, and removal of DNA by treatment with pancreatic DNase, shows a size distribution of 4 S for poly(A-U) and 4 to 10 S for RNA synthesized from nicked colicin E1 DNA. Formation of RNA covalently attached to nicked colicin E1 DNA is favored by the absence of the σ subunit of RNA polymerase, relatively low concentration of a single ribonucleoside triphosphate (20 to 50 µm), short incubation periods at 37° (less than 5 min), and a high ratio of DNA to RNA polymerase. While RNA polymerase holoenzyme catalyzes the synthesis predominantly of free RNA, the core enzyme preparations, depleted of σ subunit, synthesize predominantly covalently attached RNA at 3'-hydroxyl termini of DNA. Core enzyme preparations are also capable of initiating free RNA synthesis to a limited extent.