REGULATION OD HEME BIOSYNTHESIS IN E COLI

描述（改编自应用）：由于中心作用血红素 和siroheme在有氧和无氧呼吸中起作用， 氨基酸、过氧化物解毒、亚硝酸盐和硫酸盐 利用，血红素的生物合成需要精细的调节。在 大肠杆菌，氨基乙酰丙酸（ALA），第一个致力于 途径中的中间体，在三个步骤中合成， 谷氨酸盐在参与ALA合成的三个基因中， 最可能发生在hemA，谷氨酰-tRNA的结构基因 还原酶（GTR）。hemA与prfA共有一个启动子， 合成释放因子1。因此，对hemA的调节也必须 解释prfA的适当表达。PI的既往研究 其他研究表明hemA的表达是复杂的。是 不仅在转录起始时被调节，而且 在转录后步骤受到调节。为了访问 在这些步骤中，一系列操纵子和蛋白质融合， 将构建lacZ。三种操纵子融合体携带不同的 将构建hemA上游区的长度。对应 对于每个操纵子融合体，使用短蛋白融合体（最多10个密码子）。 hemA翻译序列）和长蛋白融合体（多达270个密码子） 将被制造。O2、生长阶段、生长速率和血红素的影响 将在携带这些基因的菌株中分析对hemA调节的限制。 融合GTR的稳定性和半衰期也将在 在O2、生长期、生长速率和血红素条件下生长的细胞 限制.这些研究将使研究人员能够确定， 一个系统的方法，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 将在rrpoH和rpoS突变体中进行转录，以评估 第三个hemA启动子的活性。在另一项研究中， 突变将被引入hemA中，以分析其在 细胞，并确定其作用，如果有的话，在hemA调节。隔离 并进行卟啉高产突变体的表征 试图找出影响血红素调节的基因 通路

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.