Initiation of transcription at phage T4 late promoters with purified RNA polymerase

Abstract

We have previously identified T4 late promoters governing the in vivo expresson of T4 late genes 23 and 24 ( P 23 and P 24). T4 late transcription in vivo is known to involve the binding of at least five phagecoded proteins to the bacterial RNA polymerase and normally requires concurrent DNA replication for DNA template activation. We show here that in vitro transcription, primarily of plasmids carrying T4 genes 23 and 24, by RNA polymerase purified from Escherichia coli at late times after T4 infection allows specific initiation at P 23 and P 24 in the absence of DNA replication. These promoters are not utilized by E. coli RNA polymerase holoenzyme, by RNA polymerase core, or by T4-modified RNA polymerase purified from cells infected with a T4 gene 55 mutant (gene 55 codes for an RNA polymerase binding protein required for late transcription). The utilization of P 23 and P 24 in vitro is sharply inhibited by NaCI concentrations greater than 100 mM, and this inhibition is partly reversed by the addition of 10% DMSO. Relaxation of plasmid DNA containing P 23 (with topoisomerase I) reduces P 23 utilization at low salt (50 mM Na +) and nearly abolishes it at high salt (250 mM Na +). P 23 utilization is discernible in linear, glucosylated hydroxymethylcytosine-containing T4 virion DNA.