Transcription from the lambda pRE promoter is activated by the lambda cII protein, which recognizes a specific binding site in the -35 region of the promoter and facilitates recognition by RNA polymerase. Preliminary evidence indicates that cII protein, in addition to its role in activating transcription from pRE, also negatively regulates its own expression by binding to its own mRNA and inhibiting its trannslation.
The first aim of the current application is to demonstrate convincingly that cII protein is a specific RNA as well as DNA binding protein. This will be done by genetic means, primarily through contruction and use of cII/lacZ fusion plasmids, and biochemically. Using cII/lacZ fusion plasmids and the many known mutations in the hypothesized translational control region, it should be possible to establish the sequence determinants on the mRNA for specific binding by cII protein. By using cII protein variants with different affinities for DNA and mRNA binding sites, it should also be possible to learn how the primary sequence of the protein influences its relative affinities for DNA and mRNA. Expression of the cII gene is inhibited when a multicopy plasmid provides """"""""OOP"""""""" RNA, a 77 base lambda transcript of unknown function. The role of """"""""OOP"""""""" RNA in translational control of cII gene expression will be investigated. The lambda cII protein is a member of a large family of proteins which regulate gene activity through binding to DNA, and the finding that cII protein is also a regulatory protein at the level of RNA would raise the question as to whether many other DNA binding proteins act in the same way. Comparison of the mode of protein-DNA recognition to protein-RNA recognition should yield interesting insights into the mechanisms of protein-nucleic acid interactions. A genetic study of cII function is also proposed, including the isolation and characterization of mutations which confer increased affinity of cII protein for its DNA binding site, mutations with increased affinities for altered promoters, and mutations with increased cross-reactivities with pRE promoters from the related bacteriophages 21 and P22.
