Utilization of promoter and terminator sites on bacteriophage T7 DNA by RNA polymerases from a variety of bacterial orders

Abstract

The transcriptional properties of bacterial RNA polymerases purified from seven different species and representing a variety of bacterial orders have been studied using the well characterized DNA from phage T7 as template. The subunit composition of the different preparations suggests that each RNA polymerase holoenzyme has a promoter structure (betabeta'alpha2sigma) similar to that of the well studied E. coli and B. subtilis enzymes. Each enzyme utilizes DNA from bacteriophage T7 as an effective template for RNA synthesis, although all preparations contain a substantial fraction of inactive enzyme. Electrophoretic analysis of the RNA products made with the different RNA polymerases in vitro using T7 DNA (deletion mutant deltaD111) as template reveals that with minor exceptions, all of the heterologous RNA polymerases utilize the same collection of promoter sites on T7 used by the E. coli host enzyme, and only those promoter sites. The T7 early terminator is also efficiently utilized by each enzyme. Since the different bacterial species from which the RNA polymerases were derived are genetically quite distant, it appears that there is a structural element in the promoter which governs its recognition and which is universally recognized among RNA polymerases of different bacterial species. While the different bacterial RNA polymerases generally utilize the same set of T7 promoter sites, the efficiency of utilization of the different promoters varies considerably for different RNA polymerases and for different reaction conditions with the same RNA polymerase. Hence although the several T7 promoter sites share the ability to be recognized by bacterial RNA polymerases, each shows a unique pattern of utilization and therefore must possess a unique element of promoter structure as well. It has previously been shown (Stahl and Chamberlin, 1977) that T7 promoters A1, C, D and E interact differently with E. coli RNA polymerase as judged by the properties of complexes formed between each promoter and the latter enzyme. Since competition takes place among different promoter sites on a template and since these sites can differ functionally, small changes in reaction conditions or in the structure of the RNA polymerase can lead to significant changes in the rate of utilization of different promoter sites even when these promoter sites share common elements. Because the T7 promoters and terminator are utilized efficiently by such a wide range of RNA polymerases and because each of the several T7 promoters possesses unique properties which govern its utilization by RNA polymerase, analysis of the transcripts formed on a T7 DNA template provides a simple and rapid procedure for detecting and analyzing alterations in bacterial RNA polymerases which affect promoter or terminator recognition or utilization.