HEME BIOSYNTHESIS--REGULATION AND CHARACTERIZATION OF GLUTAMYL TRNA

血红素生物合成——谷氨酰 TRNA 的调控和表征

• 批准号: 6107254
• 负责人: CHARLOTTE S RUSSEL
• 金额: $ 2.57万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107254
• 关键词: 5 aminolevulinate synthase; Escherichia coli; aminohydrolases; complement pathway; enzyme activity; enzyme biosynthesis; gel filtration chromatography; gene expression; genetic regulation; genetic transcription; genetic translation; glutamyltransferase; heme; molecular cloning; porphobilinogen; porphyrin biosynthesis; protein structure function

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.

描述（改编自申请）：5-氨基乙酰丙酸 (ALA) 是四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、古细菌中， 在绿色植物中，ALA是通过C5途径合成的。谷氨酰-tRNA- 合成酶 (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)，通过氨基转移酶转化为 ALA。因为葡萄糖 tRNA还用于蛋白质合成，GSA可转化为ALA 非酶促地，GTR 步骤很可能调节 ALA 合成以及整个血红素生物合成途径。因此， hemA表达调控及其5'上游的作用 不同生长条件下的区域，以及特性和 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节的研究相关。 PI 提出以下建议： (1)(a) 最大化 GTR 合成和 促进其分离、表达载体、hemA 插入物和宿主 将优化菌株（将测试 RNase 缺陷菌株 看看它是否比现在使用的菌株 HU227 有所改进）； (b) GTR 将表征为 (a) 原生大小，(b) GTR 是否形成 home- 或异源低聚物（以及后者的组成），（c）GTR 底物和抑制剂概况，(d) GTR 是否与 GTS 结合（两个 将使用杂交酵母系统，(e)GTR是否结合血红素。 (3) 通过 将 hemA 5' 上游区域（带或不带 tem 环）置于 报告基因的前面，而 ​​hemA（没有其 5' 上游区域）是 从诱导型启动子表达，将确定 GTR 是否 自我调节其合成以及是否涉及茎环。 (4) 相同的实验，但使用 gltX 5' 上游区域，以及 没有茎环，并且 hemA 在诱导型启动子前面，将 确定GTR是否调节GTS合成；使用 hemA 5' 上游区域，有或没有茎环，以及前面的 gltX 诱导型启动子，将决定 GT 是否调节 GTR 合成。