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 pentose phosphate pathway will be studied: gnd, which encodes 6-phosphogluconate dehydrogenase, and zwf, which encodes glucose 6-phosphate dehydrogenase. Growth rate dependent regulation of gnd expression is at the level of translational efficiency and involves the """"""""internal complementary sequence (ICS)"""""""", a negative control site within the coding sequence that is highly complementary to the ribosome binding site (RBS). To provide evidence for the formation of a long-range mRNA secondary structure composed of the ICS and the RBS, gnd-kan protein fusion strains will be prepared and regulatory mutants will be selected and characterized; in addition, a dedicated ribosome system will be used to demonstrate formation of the secondary structure and the role of ribosome concentration in regulation. The effect of growth rate and regulatory mutations on the single-strandedness of the RBS and ICS will be determined by methylation of in vivo RNA and subsequent analysis by transcription-amplified primer extension (TAPE), a method to be developed for this for this purpose. A phage T7 RNA polymerase expression system will be used to determine the role of transcription-translation coupling in the regulation. Additional mutations of the gnd mRNA leader will be prepared and characterized, in order to determine why its secondary structure appears to be required for the normal level of expression and for growth rate dependent regulation. Also, genetic selections will be carried out to determine whether leader function depends on a binding factor, and structure-function studies of the leader will be conducted by the TAPE method. The mechanism for growth rate dependent regulation of zwf expression will be determined by the selection and characterization of regulatory mutants with zwf-kan protein fusion strains and by the preparation of a set of deletion and base-substitution mutations in the upstream regulatory region. The mutations will also be used to identify the cis-acting site for induction of zwf expression by superoxide free radicals. The potential role of ppGpp in growth rate regulation of zwf will be determined with a plasmid strain that allows the level of the nucleotide to be varied and with a mutant that is devoid of the nucleotide.
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