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.