REGULATION OD HEME BIOSYNTHESIS IN E COLI

描述(改编自应用程序)：由于血红素的中心作用 和含铁血红素在有氧和无氧呼吸中发挥作用，生物合成 氨基酸，过氧化氢解毒，亚硝酸盐和硫酸盐 利用，血红素的生物合成需要精致的调节。在……里面 大肠埃希氏菌，氨基乙酰丙酸(ALA)，首次提出 途径中的中间体，分三步合成，从 含有谷氨酸。在参与丙氨酸合成的三个基因中，调节 最有可能发生在HEMA，谷氨酰tRNA的结构基因 还原酶(GTR)。HEMA与编码蛋白质的PrFA共享一个启动子 合成释放因子1。因此，HEMA的调节也必须 考虑到PRFA的适当表达。PI之前的研究 另一些研究表明，HEMA的表达是复杂的。它是 不仅在转录启动时被调控，而且在转录时也被 在转录后阶段受到监管。为了访问 在这些步骤中，一系列操纵子和蛋白质的融合 Lacz将被建造。三个操纵子融合携带着不同的 将建设河马上游地区的一段路段。对应 对于每个操纵子融合，一个短的蛋白质融合(最多10个密码子的 HEMA翻译序列)和长蛋白质融合(最多270个密码子) 都会被制造出来。氧气、生长阶段、生长速度和血红素的影响 对HEMA调节的限制将在携带这些病毒的菌株中进行分析 融合。GTR的稳定性和半衰期也将在#年进行研究。 在氧气、生长阶段、生长速度和血红素条件下生长的细胞 限制。这些研究将使调查人员能够确定 一种系统的方法，调节HEMA表达的因素和 它们发挥作用的步骤。引物法的推广与分析 将在rrpoH和rpos突变体中进行转录以评估 可能的第三个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，并确定其在HEMA调节中的作用。隔离 并将进行卟啉高产突变株的鉴定 试图确定影响血红素调节的基因 路径。

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.