REGULATION OF PYRIMIDINE GENE EXPRESSION IN BACTERIA

本研究的目的是阐明其调控机制， 嘧啶基因在大肠杆菌和鼠伤寒沙门氏菌中的表达。 在这些细菌中，UMP（所有物质的前体）的从头合成 嘧啶核苷酸，由六种酶催化，这些酶由六种未连接的 基因和操纵子。 这些基因和操纵子不协调地表达 并且受到尿苷或胞苷的负调节 核苷酸 所有的核苷酸效应子的确切身份 除了pyrBI操纵子以外的基因是未知的。 本研究中的实验旨在鉴定调控元件， 以测试Pyr基因表达的可能控制机制。 特别 感兴趣的是E.大肠杆菌，它编码的亚基的 嘧啶生物合成酶天冬氨酸转氨甲酰酶。 表达 这个操纵子的负调控UTP。 初步研究表明， 揭示了紧接在结构基因之前的衰减子， 在两个pyrBI启动子的任一个上游起始的位点转录是 有效终止。 确定的其他特征表明， 调控中UTP依赖性转录的相对速率 在pyrBI前导区和前导区的偶联翻译内 转录控制转录终止在衰减。 这 将通过体外定点诱变来改变DNA， 序列明显参与衰减控制。 成效为何 将在体内研究突变对调节的影响。 额外的pyrBI 调节突变将在体内分离并表征。 影响减毒频率的生理和遗传因素 和通读转录将被检查。 还包括研究 pyrC和pyrF在E.杆菌 初步研究表明， pyrF表达，其也受尿苷 核苷酸，可能不受衰减机制控制。 这些 研究将扩大。 可能与调控DNA有关 将探索结合因子。 pyrC基因作为一种 代表由胞苷调控的pyr基因 核苷酸 研究被描述为识别衰减的特征 控制和其他类型的控制机制。 UTP、CTP和 ppGpp对pyrC和pyrF表达的影响将在体外实验中测量。 依赖DNA的转录-翻译偶联系统。

The purpose of this research is to elucidate the mechanisms regulating pyrimidine gene expression in Escherichia coli and Salmonella typhimurium. In these bacteria, the de novo synthesis of UMP, the precursor of all pyrimidine nucleotides, is catalyzed by six enzymes encoded by six unlinked genes and operons. These genes and operons are expressed noncoordinately and are subject to negative regulation by either a uridine or cytidine nucleotide. The exact identities of the nucleotide effectors for all the genes except the pyrBI operon are unknown. Experiments in this study are designed to identify regulatory elements and to test possible control mechanisms of pyr gene expression. Of particular interest is the pyrBI operon of E. coli, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase. The expression of this operon is negatively regulated by UTP. Initial studies have revealed an attenuator immediately preceding the structural genes at which site transcription initiated upstream at either of two pyrBI promoters is efficiently terminated. Additional features identified suggest a model for regulation in which the relative rates of UTP-dependent transcription within the pyrBI leader region and coupled translation of the leader transcript control transcriptional termination at the attenuator. This model will be tested by in vitro site-directed mutagenesis to alter DNA sequences apparently involved in attenuation control. The effects of these mutations on regulation will be studied in vivo. Additional pyrBI regulatory mutations will be isolated in vivo and characterized. Physiological and genetic factors influencing the frequency of attenuated and readthrough transcription will be examined. Also included are studies of pyrC and pyrF expression in E. coli. Preliminary studies indicate that pyrF expression, which also is negatively regulated by a uridine nucleotide, may not be controlled by an attenuation mechanism. These studies will be extended. The possible involvement of a regulatory DNA binding factor will be explored. The pyrC gene is included as a representative of the pyr genes that are regulated by a cytidine nucleotide. Studies are described to identify features of attenuation control and other types of control mechanisms. The effect of UTP, CTP, and ppGpp on pyrC and pyrF expression will be measured in an in vitro DNA-dependent, coupled transcription-translation system.