The role of replication proteins in the regulation of bacteriophage T4 transcription. II. Gene 45 and late transcription uncoupled from replication

In this paper we have further analyzed the role of individual replication proteins in phage T4 late gene expression. One special objective has been to determine whether individual replication gene products affect late gene expression that has independently been uncoupled from replication. In the experiments which achieve this objective, T4 pol_ts ^post- lig_am ^post- exof_am ^post- amX phage have been constructed, with mutations X in the various replication genes. Viral RNA and proteins have been analyzed by hybridization-competition and by acrylamide gel electrophoresis respectively. Mutations in the replication genes have been found to produce two kinds of effects: (1) a mutation in gene 45 almost completely abolishes late gene expression. Only the gene 45 mutation does this. Gene 45 protein is continuously required for late transcription. (2) Amber mutations in the other replication genes increase the temperature sensitivity of replication-independent late T4 transcription to varying degrees. The product of gene 45 is thus identified as a third essential component of late transcription, together with the previously identified gene 33 and 55 proteins. The endonucleolytic cleavage of parental DNA in the absence of phage DNA replication has also been examined. The distribution of single-strand breaks between the complementary DNA strands is not appreciably asymmetric. Breaks are introduced into the parental DNA of all phage mutants examined, in which no DNA replication occurs, including a mutant in gene 45. In the closing discussion we attempt to analyze the processing of T4 DNA for selective gene expression. The possibility that this processing and late transcription involve a complex of many replication proteins is examined.