Interaction of RNA Polymerase from Escherichia coli with DNA. Effect of Temperature and Ionic Strength on Selection of T7 DNA Early Promoters

Abstract

Factors influencing promotor site selection by E. coli RNA polymerase were investigated using [phage] T7 DNA as template. The utilization of the 3 major early promotors. A1, A2 and A3 was followed by processing the RNA transcripts with RNAase III, which generates 3 corresponding initiator RNA fragments. The 3 promoters were functionally distinct. A strong differential effect of temperature and ionic strength on promoter selection was observed. The transition temperature of promotors A1, A2 and A3 was measured directly by preincubating, at different temperatures, RNA polymerase and T7 DNA with primer-substrate combinations which selected each site independently. The transition temperature of the 3 sites was markedly different. Promoter A3 was used predominantly at low temperature; A1 or A2 promoters were gradually activated by increasing the temperature. The temperature response curves were strongly dependent upon the salt concentration. Challenge experiments with rifampicin or poly I showed that, once preinitiation complexes are formed by incubating RNA polymerase with DNA at 37.degree. C, the 3 sites A1, 2 and A3 promote chain initiation with equal efficiency and at the same rate.