Our longterm objective is to understand key regulatory controls that operate during the processing and translation of messenger RNAs in Escherichia coli. Current work focuses on the mechanisms used in the major transcription unit of the filamentous phage to achieve the carefully balanced pattern of gene expression that permits a persistent infection in its hosts to be maintained. First, in the gene pair provided by f1 genes V and VII, the basic hypothesis for translational coupling will be tested, with emphasis on demonstrating the mechanism in vivo. The approach is to use a 16S rRNA mutant with an altered anti-Shine-Dalgarno sequence to direct entry of a spectinomycin-resistant ribosome selectively to the upstream gene, which has been provided a Shine-Dalgarno sequence complementary to the mutant 16S rRNA. Gene VII is ideal for the studies since we have shown it to be inherently inactive when present alone. Second, since we have obtained evidence that the pair of in-frame overlapping genes at the proximal end of the transcription unit is regulated both by as yet unknown rne-dependent cleavages and by translational control, the basis for their regulation will be defined further. This pair of genes is more generally representative of a largely unexplored strategy in prokaryotes which achieves the same function as alternative splicing in eukaryotes, production of two or more proteins of different size from the same gene. Third, a very strong translation initiation site discovered in the related phage IKe will be explored as a promising candidate for one that functions primarily through translational enhancers. The longterm rationale for this aim is to define as yet unidentified mRNA-rRNA interactions that mediate initiation complex assembly and thereby contribute to current efforts to refine the structure of 16S rRNA.
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