CONTROL OF ARG-2 GENE EXPRESSION IN NEUROSPORA

描述：研究的长期目标是了解 不同调控途径相互作用的分子机制 粗面脉孢菌Arg-2基因的表达调控。Arg-2基因 编码线粒体定位的氨基甲酰磷酸小亚基 合成酶。该应用程序的主要目标是理解 精氨酸特异的负性翻译调控机制 Arg-2基因的上游开放阅读框(UORF)。Arg-2 uORF是 对精氨酸可获得性作出反应的翻译调节至关重要。 预测的uORF多肽的一级氨基酸序列，即 在其他uORF中保守，对控制至关重要。精氨酸特异性 通过Arg-2 uORF进行的翻译控制将通过以下方法在体内进行评估 分析RNA和多肽合成的水平，并通过检查 核糖体在mRNA上的分布。精氨酸特异性翻译调控 将在体外使用粗毛拟青霉细胞自由翻译提取物进行评估 用合成的核糖核酸编程。 有三个具体目标。首先，反式作用基因是 对翻译控制的重要性将被确定。突变体表现出 对精氨酸特异性调节的影响将被分离和表征。 这些基因将被放入互补组，绘制图谱，并进行分离。 基因产物在精氨酸特异性翻译调控中的作用 将会被确定。第二，将使用定点突变来 确定翻译控制的序列要求。这些特点 对翻译控制很重要的uORF序列和RNA 将会被确定。第三，精氨酸特异性翻译的机制 控制权将被确定。合成uORF多肽的能力 对翻译中影响翻译过程的因素进行分析。表格的形式 产生无控制精氨酸的精氨酸，精氨酸是一种精氨酸代谢产物，或者 带电的tRNA-将被确定。

DESCRIPTION: The long term goal of the research is to understand the molecular mechanisms by which different regulatory pathways interact to control expression of the Neurospora crassa arg-2 gene. The arg-2 gene encodes the mitochondrially localized small subunit of carbamoyl phosphate synthetase. The major goal of the application is to understand the mechanism of arginine-specific, negative translational control mediated by an upstream open reading frame (uORF) in arg-2 mRNA. The arg-2 uORF is essential for translational regulation in response to arginine availability. The primary amino acid sequence of the predicted uORF peptide, which is conserved in other uORFs, is critical for control. Arginine specific translational control through the arg-2 uORF will be assessed in vivo by analyzing levels of RNA and polypeptide synthesis and by examining the distribution of ribosomes on mRNA. Arginine specific translational control will be assessed in vitro using N. crassa cell free translation extracts programmed with synthetic RNAs. There are three specific aims. First, the trans-acting genes that are important for translational control will be identified. Mutants that show effects on arginine-specific regulation will be isolated and characterized. The genes will be placed into complementation groups, mapped, and isolated. The roles of the gene products in arginine-specific translational control will be determined. Second, site-specific mutagenesis will be used to determine the sequence requirements for translational control. The features of both the uORF sequence and the RNA important for translational control will be determined. Third, the mechanism of arginine-specific translational control will be determined. The ability of a synthetic uORF peptide to affect translational processes in trans will be analyzed. The form of arginine that elicits control-free arginine, an arginine metabolite, or charged tRNA-will be determined.