It is proposed to learn that molecular basis for structure/function of the lambdoid bacteriophage N proteins, essential for antitemination of transcription by RNA polymerase of their host, Escherichia coli. These small (ca. 100 amino acid), positively charged proteins are already known by DNA (thence protein) sequence and mutations. N of phage has been shown by others to participate in a transcription complex with host RNA polymerase and 4 host 'Nus' proteins, a complex induced in response to the specific recognition of N both for NusA protein and for an RNA stem/loop structure (boxB) in the transcript of affected operons. N proteins from different phages appear to have the same impact, although the amino acid sequences of different N's seem unrelated except for an arginine-rich region near the amino terminal of each; the Arg-rich region proves critical to N interaction with boxB, but does not account for the specificity of that interaction. Directed mutation of both boxB and N from different lambdoid phages, in conjunction with available NusA variants, will be pursed in order to find the basis, for observed functional specificity. Furthermore, purified segments of N protein, now available through cloning as fusions to glutathione-S-transferase, will be tested for affinity to boxB RNA, to NusA or both in combination. Antibodies against those N segments will provide yet another specific reagent, testable against each affinity. If segments affinities emerge, mutation will be directed towards pinpointing the basis. The peculiar capacity of lambdoid bacteriophages to overcome the ability of host RNA polymerase to terminate transcription thus becomes an opportunity to explore the molecular basis of specificity between proteins and RNA. Such interactions are known to be critical, example, to the functions of HIV viruses. The simpler lambdoid components, have through cloning, all been made accessible to directed mutagenesis and easy detection of mutational impact. The major divergence of the lambdoid proteins is of further interest for considerations both of protein structure/function as well as of evolution. %%% Bacteria can be infected by viruses which take over control of the replicative machinery of the bacterial host cell for replication of the virus. This proposal focuses one a central aspect of this take-over process: copying of the viral rather that host cell DNA into messenger RNAs (which are then translated into viral proteins). A single viral protein, after recognizing a specific DNA sequence in the virus genome, complexes with host replicative proteins, thereby recruiting them to copying of viral genes. This proposal examines in detail the molecular parameters governing the protein-DNA and protein-protein interactions which result in virus take-over of the cell.
