EFFECT OF ALKYLATING-AGENTS ON INITIATION AND ELONGATION OF THE LAC UV5 PROMOTER

DNA containing the lac UV5 promoter was alkylated using bifunctional s ulfur and nitrogen mustards and a monofunctional sulfur mustard. The a lkylation sites were mapped using Taq polymerase, and the effect of al kylation on the formation of the DNA-RNA polymerase complex was determ ined using gel retardation. Alkylation was observed at all G residues in the template strand. Exposure of the alkylated DNA to Escherichia c oli RNA polymerase resulted in the formation of a DNA-enzyme complex t hat was more stable, prior to initiation, than the complex formed with nonalkylated DNA. The DNA-RNA polymerase complex formed with the alky lated DNA also demonstrated decreased ability to progress along the fu ll length of the DNA template. These observations show that, in additi on to inducing transcriptional blockages, mustards also influence the interaction between RNA polymerase and its promoter. The ability to in terfere with protein-DNA interactions may contribute significantly to the effects of these compounds in eukaryotic systems with their comple x array of transcription factors.