Utilization of 5,6-dihydrouridine 5′-triphosphate in the reaction catalyzed by Escherichia coli RNA polymerase

Abstract

1. Characteristics of the incorporation of 5,6-dihydrouridine 5′-monophosphate (H 2UMP) into RNA by DNA-dependent RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) from Escherichia coli were investigated. 5,6-Dihydro-UTP (H 2UTP) replaces UTP in the polymerase reaction at a low efficiency with dAT copolymer or DNA from salmon sperm, calf thymus and pea bud as primers. UTP stimulates H 2UMP incorporation into RNA in the presence of each of the primers when it is added to reaction mixtures during the initial stages of RNA synthesis or when RNA synthesis has leveled off. H 2UMP is incorporated into RNA next to each of the 4 common nucleotides. As compared to the pattern of UMP incorporation, it occurs less frequently next to itself or UMP and more frequently next to GMP and AMP. The molecular size of RNA containing H 2UMP is smaller than that containing UMP. These results may be related to the fact that H 2UTP is a specific but inefficient substitute for UTP in the polymerase reaction. With H 2UTP as substrate, elongation of RNA chains are retarded at positions requiring UTP participation resulting in relatively short polynucleotides which represent a partial transcription of the DNA primers. Addition of UTP permits lengthening of the polynucleotide molecules resulting in a net increase in acid insoluble polymer containing H 2UMP. 2. The conversion of H 2UMP to H 2UTP by a soluble enzyme preparation from Ehrlich ascites cells is described. Since all of the reactions, including the interconversion of UMP and H 2UMP, have been demonstrated, it is proposed that synthesis de novo of naturally occurring polynucleotide H 2UMP is a possible metabolic pathway in vivo. However, the results do not exclude the possibility that hydrogenation of specific UMP residues may occur at the polynucleotide level.