Bacteriophage T4 late transcription from plasmid templates is enhanced by negative supercoiling

Abstract

Concurrent viral replication is normally required to activate bacteriophage T4 late promoters; replication is thought to provide a template structure which is competent for late transcription. Transcription from plasmid-borne T4 late promoters, however, is independent of replication in vivo and in vitro. In this work, we have shown that, when the late gene 23 promoter is located on a plasmid, its utilization in vivo depends upon the ability of host DNA gyrase to maintain some degree of negative superhelicity. This suggests that an alternative pathway exists for activation of late promoters: DNA which is under sufficient negative torsional stress is already competent for late transcription. We also describe a method for isolating ternary complexes of plasmid DNA, RNA polymerase, and nascent RNA which have initiated transcription in vivo. The topoisomer distribution of such ternary complexes prepared from T4-infected cells showed that, late in infection, transcriptional activity resides primarily in the subset of the plasmid population with the most negatively supercoiled topoisomers. However, the overall transcriptional pattern in these ternary complexes indicated that both vector and T4 sequences are actively transcribed. Much of this transcriptional activity could be independent of gp55, the T4-specific RNA polymerase-binding protein that confers late promoter recognition.During infection of Escherichia coli by bacteriophage T4, a program of modifications to the host RNA polymerase generates three major temporal classes of phage transcripts (reviewed in references 7, 15, and 42). Late transcription requires the products of T4 genes 33, 45, and 55. Gene product 55 (gp55) confers specificity for late T4 promoters on RNA polymerase core (26). The roles of gp33 and gp45 in transcription remain unclear, but gp45 is known also to play a role in DNA replication as an accessory protein to the replication complex (1). Late promoters share the sequence TATAAATA centered at about position -10 from the start of transcription, but require no position -35 sequence (12,14).Late transcription is also distinguished by a requirement for concurrent viral DNA replication (45). Late transcription can be uncoupled from replication in genetic backgrounds which are thought to stabilize nicks in the DNA. These observations led to the hypothesis that late transcription requires a competent template which has been activated by DNA replication (46). Uncoupling can also be achieved by placing the promoter on a plasmid template (21, 23). Late transcription from plasmid templates in vivo is otherwise faithful in its initiation sites and in regulation by genes 33, 45, and 55 (14, 23). Additionally, supercoiled plasmids are efficient templates for in vitro transcription with purified T4 late RNA polymerase (24). Therefore it appears that plasmid templates are intrinsically competent for late transcription.The topoisomer profile of plasmids changes drastically after T4 infection. We have argued that there are two reasons for this topoisomerizat...