Transcription from a bacteriophage T4 middle promoter using T4 motA protein and phage-modified RNA polymerase

Abstract

The bacteriophage T4 motA protein is required for transcription from T4 middle promoters. These promoters, which contain the Escherichia coli promoter consensus sequence at the -10 region (TATAAT) but a unique sequence centered at -30 ((a/t)(a/t)TGCTT(t/c)A) (Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T., and Gold, L. (1988) J. Mol. Biol. 199, 241-258), become active about 2 min after infection, a time when the host RNA polymerase has been modified by phage proteins. This paper shows that motA protein binds to a T4 middle promoter in vitro and that the addition of the motA protein allows in vitro transcription from this promoter by T4-modified RNA polymerase. The T4 motA gene was cloned into a multicopy plasmid that complemented T4 motA mutants in vivo. MotA protein, partially purified from cells containing a motA+ plasmid, specifically retarded the electrophoretic mobility of an oligomer containing the T4 middle promoter located 195 bases upstream of uvsX (PuvsX). RNA polymerase isolated from infected cells during T4 middle gene expression supported in vitro transcription from PuvsX only when fractions containing the motA protein were added. In contrast, unmodified host RNA polymerase catalyzed the synthesis of minor amounts of RNA from PuvsX, but this synthesis was not motA dependent. Thus, the in vitro transcription system described here provides the basis for a detailed study of the phage and host factors needed to regulate T4 middle gene expression.