Our studies have importance towards a general understanding of the regulation of gene expression. Our immediate goal is to investigate some specific features of the transcription of the lac genes of Escherichia coli and their relationship to messenger decay. The lac operon consists of three genes, Z, Y and A, extending over about 5100 base pairs (bp). The lac genes are transcribed polycistronically and the transcript includes short leading and trailing sequences as well as significant intercistronic regions. In recent work, we have located the presumptive operon terminator beyond lacA, and have shown that a fraction of the lac transcripts ends at a discrete site in the Z-Y space, coincident with an apparent rho-independent terminator. Our results indicate that this terminator is the basis in large part for the natural polarty of lac gene expression. In addition, others have suggested that this region possesses an RNase III or other, processing site, implied to play a role in the decay of the messenger. To more clearly define the function of the 54 bp lac Z-Y intergenic region as a determinant of polarity and messenger decay, and to explore the mechanisms associated with these processes, sequences in the lac Z-Y region have been altered by recombinant DNA techniques and moved into the context of a galK expression plasmid in which distal gene expression can be readily quantitated. These constructions will be tested in vivo and in vitro to characterize messenger transcription and decay, RNA processing and polarity effects. These constructions are uniquely organized because they contain a functional mini-lacZ gene, arranged so that the full length Z gene can be readily restructured. Their use will facilitate the manipulation of the 5'- leader and 3'-lacZ regions, as well as separate the function that these regions play from those of the internal sequences. Finally, in comparison studies, a new method developed in the lab will be used to probe for alterations at the 5' end of the lacZ messenger during its functional decay, and the presumptive rho- dependent termination site distal to the operon will be characterized in vitro.
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