REGULATION OF CARAB IN PSEUDOMONAS AERUGINOSA

第一个目的是阐明间隔区的功能 来自铜绿假单胞菌的carA序列。 比较 具有氨基末端氨基酸的carA的衍生氨基酸序列 氨甲酰磷酸合成酶的小亚基的序列显示， 由四个密码子指定的氨基酸残基不存在于 多肽。 这种共线性的缺乏可以解释为 在翻译或多肽剪接过程中的核糖体跳跃。 在 体内和体外追踪标记实验以及诱变 研究将区分这两种模式。 表达的研究 在异源系统中的carA将评估一个 交易因素 将采用定点诱变来 研究间隔序列的功能，并确定是否 它在表达调节中发挥作用。 突变也将是 用于识别决定核糖体的序列特征， 跳跃或多肽剪接。 结果应具有一般性 对我们理解间隔区序列的功能具有重要意义 在结构基因中。 第二个目的是阐明调节的机制， carAB表达。 定量引物延伸和诱变 实验将被用来测试的假设，多个 启动carA上游的转录物受到不同的调控， 嘧啶、精氨酸和另一种σ因子。 外贸 融合和诱变将测试这一假设， carAB涉及衰减机制。 编码一种调节基因的基因 参与控制某些基因表达的因子， 精氨酸的生物合成和有氧催化作用将通过以下方法来鉴定： 插入诱变 该基因将被克隆，表征， 产品识别。 一个长期目标是隔离监管 并研究其与控制区的相互作用。 的 鉴于资料有限，拟议的研究具有重要意义 在这种机会主义的染色体基因调控上， 病原体 还将对监管进行比较性但有限的研究 以确定是否存在非共线性与 不同的监管模式。

The first objective is elucidation of the function of the spacer sequence within carA from Pseudomonas aeruginosa. Comparison of the derived amino acid sequence of carA with the amino terminal amino acid sequence of the small subunit of carbamoylphosphate synthetase revealed that the amino acid residues specified by four codons are not present in the polypeptide. This lack of colinearity can be explained on the basis of ribosomal hopping during translation or polypeptide splicing. In vivo and in vitro chase-labeling experiments as well as mutagenesis studies will distinguish between the two models. Studies on expression of carA in heterologous systems will assess the requirement of a transacting factor. Site-directed mutagenesis will be employed to investigate the function of the spacer sequence and to determine whether it plays a role in regulation of expression. Mutagenesis will be also employed to identify the sequence features that determine ribosomal hopping or polypeptide splicing. The results should be of general significance to our understanding of the function of spacer sequences within structural genes. The second objective is elucidation of the mechanism of regulation of carAB expression. Quantitative primer extension and mutagenesis experiments will be employed to test the hypothesis that the multiple transcripts initiated upstream of carA are differentially regulated by pyrimidines, arginine, and an alternative sigma factor. Studies with fusions and mutagenesis will test the hypothesis that regulation of carAB involves an attenuation mechanism. The gene encoding a regulatory factor involved in control of expression of certain genes of biosynthesis and aerobic catabolism of arginine will be identified by insertional mutagenesis. This gene will be cloned, characterized, and its product identified. A long term goal is to isolate the regulatory product and to study its interactions with the control region. The proposed studies are of significance in view of the limited information available on regulation of chromosomal genes in this opportunistic pathogen. Comparative but limited studies on regulation will be carried out also in P. stutzeri to determine if the absence of non-colinearity relates to a different regulatory pattern.