Application): 5-Aminolevulinic acid (ALA) is the universal precursor of the tetrapyrrole pigments, including hemes, chlorophylls, and vitamin B12. In most bacteria, in archae, and in green plants, ALA is synthesized by the C5 pathway. Glutamyl-tRNA- synthase (GTS), the gene product of gltX, catalyzes the synthesis of glutamyl-tRNA (glu-tRNA), which is reduced by glutamyl semi-aldehyde (GSA), which is converted to ALA by an amino transferase. Because glu- tRNA is also used for protein synthesis, and GSA can be converted to ALA non-enzymatically, it is highly probable that the GTR step regulates ALA synthesis and consequently, the whole heme biosynthesis pathway. Thus, the regulation of expression of hemA, the roles of its 5' upstream region under different conditions of growth, and the properties and structure of GTR and its interactions with other molecules are all relevant to a study of the regulation of heme biosynthesis. The PI proposed the following: (1)(a) To maximize GTR synthesis and facilitate its isolation, the expression vector, hemA insert, and host strain will be optimized (an RNase-deficient strain will be tested to see if it is an improvement over HU227, the strain used now); (b) GTR will be characterized as to (a) native size, (b) whether GTR forms home- or hetero-oligomers (and the composition of the latter), (c) GTR substrate and inhibitor profiles, (d) whether GTR binds to GTS (the two- hybrid yeast system will be used, (e) whether GTR binds heme. (3) By placing the hemA 5' upstream region, with and without its tem loops, in front of a reporter gene while hemA (without its 5' upstream region) is expressed from an inducible promoter, it will be determined whether GTR self-regulates its synthesis and whether stem-loops are involved. (4) The same experiment, but using the gltX 5' upstream region, with and without stem-loops, and hemA in front of an inducible promoter, will determine whether GTR regulates GTS synthesis; using the hemA 5' upstream region, with and without its stem-loops, and gltX in front of an inducible promoter, will determine whether GT regulates GTR synthesis.
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