On the influence of thymidine analogues on the activity of phage fd promoters in vitro

Abstract

RF I DNA of phage fd containing 5-bromo-deoxyuridine (br⁵U_d) or deoxyuridine (U_d) instead of deoxythymidine (T_d) in the codogenic strand was synthesized in vitro. The modified genomes could be cleaved by restriction endonuclease Hpa II. Although the recognition site of Hpa II is CCGG, the cleavage rate was significantly reduced with U_d-containing DNA. Both base substitutions altered the mobilities of several DNA fragments under the conditions of polyacrylamide gel electrophoresis. The fragments containing binding sites for RNA polymerase were assayed for the rates of stable complex formation. The substitution of T_d for both, U_d and br ⁵U_d, strongly influenced this parameter. Thus the methyl group of T_d has to be regarded as one of the sites in DNA which determine the rate of stable RNA polymerase binding and thereby possibly mediate promoter activity in vitro (24, 25, 26). In most cases the rate of complex formation was decreased by U_d, but increased by br ⁵U_d.