The proposed research investigates basic mechanisms in virus- host interactions, specifically the shift from host to viral transcription, using the E. coli-bacteriophage T4 system. Emphasis is placed upon the T4 gene product alc/unf and its interaction with other factors in shutting off transcription from cytosine-containing DNA and unfolding the host nucleoid. Our recent work has established that gpalc generally inhibits transcription from dC-DNA both in vivo and in vitro, probably by promoting pausing and termination of elongating RNA transcripts. Proposed research includes: 1) Completion of cloning of the cDNA for alc into an expression vector and purification of the alc protein; 2) use of the purified protein to further study the inhibition by gpalc of RNA chain elongation; 3) investigation of the interaction of gpalc with other factors involved in transcriptional pausing and termination; 4) determination of whether gpalc acts through binding to DNA, RNA polymerase, or a complex; 5) analysis of sites on DNA where gpalc binding occurs; 6) studies of the relationship between the observed transcriptional inhibition and the other known effect of gpalc/unf - the unfolding of the host nucleoid; and 7) studies to determine what other T4 genes are also involved in shutoff of host transcription.
