ORGANIZATION AND REGULATION OF THE ATCASE CISTRONS

这项持续研究计划的长期目标是开发一种 详细了解法规和结构/功能 天冬氨酸转氨甲酰酶的关系。 顺反子编码 调节和催化多肽被组织为双顺反子操纵子 但基因表达的控制机制尚不确定： 衰减，平移耦合到次要带电物种 乙酰基-tRNA，或转录起始的反式阻遏物调节。 这些监管计划涉及以下方面的复杂相互关系： 氨基酸生物合成和从头嘧啶生物合成。 是一种理想 模型系统，用于描述对可操纵的 相关代谢物的内源性合并物。 监管机制 对照将通过突变启动子的核苷酸测序进行评价 从启动子/操纵子lacZ融合分离的突变体，通过与 pyrBI启动子从其他肠道细菌，通过创建特定的 缺失和位点特异性突变，通过在各种 反式作用突变（rpo*，pyrBI和pyrE的上启动子，以及在pyrBI和pyrE中的上启动子）。 调节内源性核苷酸库的特别设计的菌株），和 通过在异源宿主细胞中表达各种pyrBI操纵子。 此外，已知pyrBI、pyrE和pyrF包含协同结构。 调节子，其响应于尿苷酸或尿苷酸/胞苷酸池和pyrE 具有类似的衰减器结构。 另一个目标是描述 这一合作机制。 此外，很明显， 初步证据表明，基因表达调控的细节 在其他肠道细菌中，以微妙的方式变化， 对机械细节的进一步了解。 这项研究的第二个长期目标是帮助 定义所涉及的结构特征的描述 ATCase的催化和变构控制。 的核苷酸序列 调节顺反子（pyrI）已经确定了一些差异， 已发表的氨基酸序列，需要验证。 其他案件 肠道细菌具有相同的结构，2（c3）：3（r2），但在 催化和调节特性。 此外，argI 顺反子编码鸟氨酸氨甲酰转移酶的多肽， 代表了一种不同但进化上相关的酶。 将 可以通过以下方式探测酶特性中的功能变化： 比较推导的氨基酸序列和预测的二级结构。 最后，位点特异性诱变将允许操纵特异性突变。 残基

The long-term objective of this continuing research program is to develop a detailed understanding of the regulation and structure/function relationships of aspartate transcarbamoylase. The cistrons encoding the regulatory and catalytic polypeptides are organized as a bicistronic operon but the mechanism of control of gene expression is uncertain: attentuation, translational coupling to minor charged species of arginyl-tRNA, or trans repressor modulation of transcriptional initiation. These regulatory schemes involve a complex interrelationship between aminoacid biosyntheses and de novo pyrimidine biosynthesis. It is an ideal model system for describing the regulatory logic response to manipulable endogenous pools of related metabolites. The mechanisms of regulatory control will be evaluated by nucleotide sequencing of mutant promoter mutants isolated from promoter/operator lacZ fusions, by comparison with pyrBI promoters from other enteric bacteria, by the creation of specific deletions and site-specific mutations in vitro, by expression in various trans-acting mutations (rpo*, an up-promoter for pyrBI and pyrE, and in specially designed strains that modulate endogenous nucleotide pools), and by expression of various pyrBI operons in heterologous host cells. Furthermore, it is known that pyrBI, pyrE, and pyrF comprise a cooperative regulon which responds to uridylate or uridylate/cytidylate pools and pyrE has a similar attenuator structure. Another objective is to describe the mechanism of this cooperative regulation. In addition, it is clear from preliminary evidence that the details of the regulation of gene expression in other enteric bacteria is varied in subtle ways that may provide additional insight into mechanistic particulars. The second long-term objective of this research is to aid in the description of the structural characteristics involved in defining the catalytic and allosteric control of ATCase. The nucleotide sequence of the regulatory cistron (pyrI) has identified some differences from the published aminoacid sequence and need to be verified. ATCases from other enteric bacteria possess the same architecture, 2(c3):3(r2), yet differ in both catalytic and regulatory characteristics. In addition, the argI cistron encodes a polypeptide for ornithine transcarbamoylase which represents a divergent but evolutionarily related enzyme. It will be possible to probe the functional changes in enzymatic characteristics by comparing deduced aminoacid sequences and predicted secondary structures. Finally, site-specific mutagenesis will allow the manipulation of specific residues.