The long term objective is to elucidate the mechanisms of a fundamental genetic regulatory process, growth rate dependent regulation of gene expression. Two Escherichia coli genes for enzymes of the hexose monophosphate shunt will be studied: gnd, which encodes 6 phosphogluconate dehydrogenase (6PGD) and zwf, which encodes glucose 6-phosphate dehydrogenase (G6PD). Growth rate dependent regulation of gnd expression occurs at the posttranscriptional level and involves a site of negative control which lies within the coding sequence for 6PGD and which is highly complementary to the ribosome binding site (RBS). A model is proposed which suggests that the regulation involves complementary base pairing in the mRNA between the internal homology sequence (IHS) and the RBS. To test the model a series of RBS and IHS deoxyoligonucleotides containing one or more mutations at specific sites will be synthesized, cloned by a new method, and the effect of the mutations singly and in combinations will be determined for the growth rate dependent regulation of gnd-lacZ protein fusions. The level of 6PGD is growth rate uninducible in hisT mutants of Salmonella typhimurium which are defective in the tRNA modifying enzyme pseudouridine synthase I (PSUI). Genetic analysis and in vitro experiments with positive regulatory role of hisT by binding to a putative PSUI recognition site which is within the RBS-IHS hairpin and destabilizing the structure. The growth rate dependence of the amount, synthesis rate, functional and chemical half-life and utilization of gnd mRNA will be determined and the hypothesis tested that the growth rate dependent regulation of 6PGD level is effected by endonucleolytic cleavage of the MRNA at the IHS. The molecular basis for the overproduction and altered growth rate dependence of G6PD level in mutants with cis-dominant zwf mutations will be determined by cloning and DNA sequence analysis. These data and the properties of zwf-lac operon and protein fusions should show whether zwf and gnd are regulated by similar mechanisms. Achieving the project's specific aims should provide a detailed understanding of the unusual mechanism that regulates gnd expression and in knowledge of growth rate dependent regulation of non-ribosomal genes and thus bacterial growth.
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