Application): Because of the central role heme and siroheme play in aerobic and anaerobic respiration, the biosynthesis of amino acids, peroxide detoxification, and nitrite and sulfate utilization, the heme biosynthetic requires exquisite regulation. In Escherichia coli, aminolevulinic acid (ALA), the first committed intermediate in the pathway, is synthesized in three steps, starting with glutamate. Of the three genes involved in ALA synthesis, regulation most probably occurs at hemA, the structural gene for glutamyl-tRNA reductase (GTR). hemA shares a promoter with prfA, which encodes protein synthesis release factor 1. Therefore, regulation of hemA must also account for appropriate expression of prfA. Previous studies by the PI and others have shown that expression of hemA is complex. It is modulated not only at the initiation of transcription, but also is subject to regulation at a post-transcriptional step. In order to access regulation at these steps, a series of operon and protein fusions to lacZ will be constructed. Three operon fusions carrying different lengths of the hemA upstream region will be constructed. Corresponding to each operon fusion, a short protein fusion (up to 10 codons of the hemA translated sequence) and a long protein fusion (up to 270 codons) will be made. The effects of O2, growth phase, growth rate, and heme limitation on hemA regulation will be analyzed in strains carrying these fusions. The stability and half-life of GTR will also be studied in cells grown under conditions of O2, growth phase, growth rate, and heme limitation. These studies will enable the investigators to determine, in a systematic way, the factors which regulate hemA expression and the steps in which they function. Primer extension and analysis of transcription in rrpoH and rpoS mutants will be carried out to assess the activity of a putative third hemA promoter. In another study, mutations will be introduced into hemA to analyze its function in the cell, and to determine its role, if any, in hemA regulation. Isolation and characterization of porphyrin over-producing mutants will be carried out in an attempt to identify genes which affect regulation of the heme pathway.
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