We will continue on-going research on the conversion from host to bacteriophage macromolecular synthesis after infection of E. coli by phage T4. Our work still centers on the alc gene, which directs the host RNA polymerase away from dC-DNA to the HMdC-DNA of the phage, but also goes on to explore other aspects of the transition. We will continue in-vivo studies of the interaction of gpalc with the B subunit of RNA polymerase by using various polymerase mutants. A major project will be purification of gpalc from clones carrying the gene. The protein will then be used in several in vitro studies, including: (a) effects of purified gpalc on transcription of T7 DNA in vitro; (b) use of other templates to study the generality and specificity of alc-mediated inhibition; (c) interaction of gpalc with termination factors; (d) analysis of proteins bound to DNA or to transcription complexes; (e) footprinting analysis of gpalc binding to DNA alone and in the presence of polymerase. We will continue our studies on the effects of gpalc on the host nucleoid structure, using psoralen binding in vivo. We will investigate other phage-encoded proteins that may be involved in the transition from host to phage metabolism, particularly looking for alterations in the functional or chemical stability of host messenger RNA and factors that change initiation factors or that bind to host ribosomes, altering translational specificity. By cloning otherwise uncharacterized T4 immediate-early genes in tightly-regulated expression vectors, we will look for genes affecting host physiology. We will continue to update the T4 genomic map, to assemble T4 DNA sequences, and to host the semiannual international T4 meeting.
