HEME BIOSYNTHESIS--REGULATION AND CHARACTERIZATION OF GLUTAMYL TRNA

描述(改编自申请)：5-氨基乙酰丙酸(ALA) 是四聚吡咯颜料的通用前体，包括 血红素、叶绿素和维生素B12。在大多数细菌中，在古细菌中， 在绿色植物中，丙氨酸通过C5途径合成。谷氨酰-tRNA- 合酶(GTS)是gltX的基因产物，催化合成 谷氨酰-tRNA(Glu-tRNA)，被谷氨酰半醛还原 (GSA)，由氨基转移酶转化为丙氨酸。因为Glu- TRNA也用于蛋白质合成，GSA可以转化为ALA 从非酶的角度来看，GTR步骤很有可能调节ALA 合成，因此，整个血红素生物合成途径。因此， HEMA的表达调控及其5‘上游的作用 区域在不同的生长条件下，以及它们的属性和 GTR的结构及其与其他分子的相互作用都是 与血红素生物合成调控的研究有关。 PI提出了以下建议：(1)(A)最大限度地增加GTR合成和 促进其分离、表达载体、HEMA插入和宿主 将对菌株进行优化(将对RNase缺陷菌株进行测试以 看看它是否比现在使用的HU227株有所改进)；(B)Gtr 将以(A)原生大小为特征，(B)gtr是否形成家园- 或杂低聚物(以及后者的组成)，(C)Gtr 底物和抑制剂谱，(D)GTR是否与GTS结合(这两个- 将使用混合酵母系统，(E)GTR是否与血红素结合。(3)由 将HEMA 5‘上游区域(带和不带温度环)放置在 而HEMA(没有其5‘上游区)是报告基因的前端 从可诱导启动子中表达，将确定GTR是否 自我调节其合成以及是否涉及茎环。(4) 同样的实验，但使用的是gltX 5‘上游区域，带有和 没有茎环，赫马在一个可诱导的启动子面前，将 确定GTR是否调节GTS合成；使用HEMA 5‘ 上游区域，带和不带其茎环，以及位于 一个可诱导的启动子，将决定GT是否调节GTR 综合。

Description (Adapted from the 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.