Template properties of polyribonucleotides containing uracil or modified uracil in the RNA polymerase reaction

Abstract

The kinetic characteristics of the Micrococcus lysodeikticus RNA polymerase were examined with model polyribonucleotide templates. Two types of reaction were observed. (1) Transcription reactions were directed by two classes of templates: (a) in the presence of ATP, untreated poly U directed the synthesis of poly A in proportion to the poly U concentration, and (b) in the presence of ATP and YTP, poly (U, X) directed the synthesis of poly (A, Y), where Y is the Watson-Crick complementary nucleotide to X. (2) Reiteration reactions were directed by two classes of templates: (a) in the presence of ATP, poly U modified by ultraviolet irradiation or photochemical reduction directed the synthesis of poly A in proportion to the ATP concentration, and (b) in the presence of ATP and the absence of YTP, poly (U, X) directed the synthesis of poly A. Reiteration was dependent on the template mole fraction of U. Reiteration reactions are directed by specific template sites in which sequences of U are terminated by non-U bases. The minimum sequence length for reiterative poly A synthesis was estimated from a statistical correlation of synthetic rates directed by families of copolymer templates containing various proportions of two nucleotides, with sequence frequencies of the type (UpUp…UpX-) 1 expected, assuming a random distribution of U- and X-containing nucleotides. A sequence of at least two U residues terminated by an X residue is required for this reiterative synthesis of poly A. Although the non-U base terminating a sequence of U residues may be a natural (A, G, or C) base, as well as 5,6-dihydrouracil or a uracil photoproduct, there is a distinction between the reactions induced by these two types of template blocks. If a sequence of U residues is terminated by a natural base, the addition of the NTP complementary to that terminating base will depress reiterative poly A synthesis and transcription to form a copolymer is observed. But if the tracts of U residues are terminated by modified uracil, either a photo-product or 5,6-dihydrouracil, there is no depression of reiterative poly A synthesis by low concentrations of any of the other three common nucleoside triphosphates.