REGULATION OF GENE EXPRESSION IN BACTERIOPHAGE T4

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After infection of E. coli by bacteriophage T4, the host RNA polymerase acquires several small phage-induced polypeptides and its alfa subunits are ADP-ribosylated. The role of these modifications in transcription control will be studied using a combination of biochemical, genetic and physiological approaches. We have purified four of the associated polypeptides (15K, 19K, 25K and 29K proteins) as well as RNA polymerases differing in the state of ADP-ribosylation and propose to study their interactions in direct binding assays. The kinetics of in vivo formation of these proteins and their interactions with other intracellular components will be analysed in order to understand the coordination between transcription and other events of T4 development such as DNA replication. We will identify T4 genes involved in the modifications and use their mutants to determine their role in phage development. In vitro experiments are proposed to study the interaction of normal and modified RNA polymerase with individual promoters with emphasis given to the change of specificity of promoter recognition from early to late sites. We have already shown that 25K protein induces late promoter specificity while 15K protein decreases the utilization of early promoters. To understand the molecular basis of the specificity change we will determine the kinetic parameters of promoter functioning with different forms of RNA polymerase. Experiments directed at biochemical identification of T4-induced antitermination mechanism are also proposed. The in vitro experiments will be backed up by the analysis of in vivo functioning of the same transcription sites using recently sequenced tRNA gene region of T4 as the experimental system.