The bacteriophage lambda cIII gene product permits lambda to enter the lysogeneic pathway by stabilizing the lambda cII regulatory protein. Several factors regulate cIII expression at the transcriptional level. It appears the also the initiation of translation of the cIII gene is subject to regulation. Analysis of a set of cIII-lacz gene and operon fusions demonstrated the presence of a sequence upstream of the cIII ribosome binding site that is required for cIII translation. This sequence contains an RNase III processing site. Expression of cIII is drastically reduced in cells lacking RNase III. Paradoxically, we found that unprocessed mRNA, but not processed RNA, can efficiently direct the translation of the cIII message. We proposed that the ribosome binding site of cIII is occluded by mRNA secondary structure. Initiation of cIII translation requires the presence of a sequence that is acted upon by RNase III. The function of RNase III is to expose the cIII ribosome binding site permitting initiation of translaton. This regulatory function of RNase III may be distinct from its action in the cleavage of mRNA; it is possible the mRNA binding per se by RNase III activates cIII translation. There are very few examples where positive translational control of gene expression is clearly demonstrated. Furthermore the laws that govern the recognition of specific RNA sites by RNA binding proteins such as RNase III are poorly understood. Genetic and biochemical approaches will be employed to study the mechanism of the regulation of cIII expression. A genetic system for the generation, selection and study of mutation in the regulatory site has been developed. Procedures for the selection of specific mutations in rnc are given. The biochemical approach includes the use of in vitro synthesized RNA that will be used for studies in the binding and processing by RNase III, in cIII translation by purified and crude translation systems, and in the characterization of mutations in rnc. Introduction of specific base alterations will be analyzed for their effects on gene expression and RNA secondary structure.
