REGULATION OD HEME BIOSYNTHESIS IN E COLI

大肠杆菌中 OD 血红素生物合成的调节

• 批准号: 6301689
• 负责人: SHARON D COSLOY
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6301689
• 关键词: 5 aminolevulinate synthase; Escherichia coli; bacterial proteins; chimeric proteins; enzyme activity; gene expression; gene mutation; genetic promoter element; genetic regulation; genetic transcription; glutamyltransferase; lac operon; porphyrin biosynthesis; posttranscriptional RNA processing; posttranslational modifications; protein sequence; protein signal sequence; structural genes

Description (Adapted from 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.

描述（改编自申请）：由于血红素的核心作用 西罗血红素在有氧和无氧呼吸、生物合成中发挥作用 氨基酸、过氧化物解毒、亚硝酸盐和硫酸盐 利用时，血红素生物合成需要精细的调节。在 大肠杆菌，氨基乙酰丙酸（ALA），第一个被承诺的 该途径的中间体分三步合成，开始 与谷氨酸。参与 ALA 合成、调节的三个基因 最有可能发生在 hemA，谷氨酰-tRNA 的结构基因 还原酶（GTR）。 hemA 与 prfA 共享一个启动子，编码蛋白质 合成释放因子1。因此，hemA的调节也必须 考虑 prfA 的适当表达。 PI 之前的研究 等人已经表明，hemA 的表达是复杂的。这是 不仅在转录起始时受到调节，而且 受转录后步骤的调控。为了访问 在这些步骤的调节，一系列操纵子和蛋白质融合 将构建 lacZ。三个操纵子融合携带不同的 将构建 hemA 上游区域的长度。相应的 每个操纵子融合，一个短的蛋白质融合（最多 10 个密码子） hemA 翻译序列）和长蛋白融合体（最多 270 个密码子） 将被制作。 O2、生长阶段、生长速率和血红素的影响 将在携带这些的菌株中分析对hemA调节的限制 融合。 GTR 的稳定性和半衰期也将在 细胞在 O2、生长期、生长速率和血红素条件下生长 局限性。这些研究将使研究人员能够确定， 系统地研究了调节hemA表达的因素和 它们发挥作用的步骤。引物延伸和分析 将进行 rrpoH 和 rpoS 突变体的转录以评估 推定的第三个hemA启动子的活性。在另一项研究中， 将突变引入hemA以分析其在 细胞，并确定其在 hemA 调节中的作用（如果有的话）。隔离 并将进行卟啉过量产生突变体的表征 试图识别影响血红素调节的基因 途径。