Changed promoter specificity and antitermination properties displayed in vitro by bacteriophage T4-modified RNA polymerase

Abstract

A 5.5 × 10 3 base-pair fragment of bacteriophage T4 DNA carrying genes 1, 3, 57, ipI and a cluster of transfer RNA genes was used as template for RNA polymerase isolated from uninfected Escherichia coli and from T4-infected bacteria. RNA transcripts were fractionated by gel electrophoresis and mapped by using as transcription template the 5.5 × 10 3 base fragment cleaved with different restriction enzymes. The comparison of the transcripts synthesized by the two RNA polymerases revealed a dramatic difference in their initiation specificities and abilities to utilize a transcription termination site. The T4-modified polymerase utilizes three new promoters on the template DNA fragment that are not utilized by the host enzyme. The modified enzyme, however, fails to produce some of the transcripts synthesized by the host RNA polymerase. The ability of T4-modified RNA polymerase to terminate transcription at a terminator present in the template DNA fragment is greatly reduced as compared to the unmodified host enzyme. The factors responsible for the new initiation and termination properties are associated with RNA polymerase core component. Analysis of RNA polymerase from bacteria infected with T4 mutants demonstrates that the new promoter specificity and the antitermination effect are caused by different factors.