CONTROL OF ARG-2 GENE EXPRESSION IN NEUROSPORA

描述：研究的长期目标是了解 不同调节途径相互作用的分子机制， 控制粗糙脉孢菌arg-2基因的表达。 arg-2基因 编码氨甲酰磷酸的一个位于细胞内的小亚基 合成酶 应用程序的主要目标是了解 介导的丝氨酸特异性负翻译控制机制 arg-2 mRNA的上游开放阅读框（uORF）。 arg-2uORF是 对于响应精氨酸可用性的翻译调节至关重要。 预测的uORF肽的一级氨基酸序列， 在其他uORF中保守的，是控制的关键。 精氨酸特异性 通过arg-2uORF的翻译控制将在体内进行评估， 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 将使用N. crassa细胞游离翻译提取物 用合成RNA编程。 有三个具体目标。 首先，反式作用基因， 重要的翻译控制将确定。 突变体显示 将分离和表征对精氨酸特异性调节的作用。 这些基因将被放置到互补组中，绘制并分离。 基因产物在甜菜碱特异性翻译调控中的作用 将被确定。 其次，将使用位点特异性诱变来 确定翻译控制的序列要求。 的特征 uORF序列和对翻译控制很重要的RNA 将被确定。 第三，对甜菜碱特异性翻译的机制进行了研究。 控制将被确定。 合成的uORF肽能够 影响翻译过程中的反式将进行分析。 的形式 精氨酸，其增强了对游离精氨酸、精氨酸代谢物的控制，或 将确定带电荷的tRNA。

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.