Interactions of a proteolytically nicked RNA polymerase of bacteriophage T7 with its promoter

Abstract

The association of nicked RNA polymerase of bacteriophage T7 (Ikeda, R. A., and Richardson, C. C. (1987) J. Biol. Chem. 262, 3790-3799) with the T7 phi 10 promoter has been examined by DNA cleavage protection. The phi 10 promoter consists of a 23-base pair consensus sequence that extends from -17 to +6 with respect to the site of the initiation of transcription (+1). Nicked T7 RNA polymerase alone protects 20 bases from -21 to -2 (+/- 1) base at each border. Initiation and synthesis of the trinucleotide r(GGG) expands and shifts the sequence protected by nicked T7 RNA polymerase. Twenty-five bases are protected from -17 to +8 (+/- 1). The polymerization of three additional ribonucleotides, synthesis of the hexamer r(GGGAGA), further expands the protected sequence. Twenty-seven bases are protected from -17-+10 (+/- 1). Finally, the synthesis of a pentadecaribonucleotide transcript, r(GGGAGACCACGG), leads to the formation of a transcription complex that protects 22 bases from -2-+20 (+/- 1). In comparison to the sequences protected by T7 RNA polymerase the sequences protected by the nicked enzyme are shortened at the 5' end and are translocated downstream much earlier during the initiation of transcription. It appears that a portion of the DNA contacts made at the amino terminus of T7 RNA polymerase are disrupted in the small fragment of nicked T7 RNA polymerase. The changes that are observed in the sequences protected by nicked T7 RNA polymerase are reflected in the physical characteristics of the DNA X enzyme complexes. The number of ion pairs formed by the r(GGG)-initiated complex of the nicked enzyme is reduced, and the association constant for the formation of the r(GGG)-initiated complex is decreased as compared to the intact T7 RNA polymerase.