Control of arg-2 Gene Expression in Neurospora

描述（由申请人提供）：这项工作的目的是了解上游开放阅读框（uORF）编码的肽如何控制mRNA上核糖体的运动并调节基因表达。精氨酸衰减肽（AAP）由指定真菌精氨酸（Arg）生物合成酶的mrna中的uORF编码，并在Arg高时降低基因表达。用真菌和小麦胚芽提取物在体外观察aap介导的调节作用。新生的AAP导致核糖体对Arg的反应停滞。停滞的核糖体阻断了进入下游启动酶合成的起始结肠的途径。AAP是一种独特的真核肽，因为它的合成可以阻止参与终止和延伸的核糖体。它的研究提供了特殊的机会来检查仍然不理解的中心翻译过程。这将为评估干扰转译的抗生素的功能提供有关核糖体内事件的宝贵信息。由于控制基因表达的uorf存在于动物、植物、真菌、病毒和细菌中，因此拟议的工作与理解这些重要的调控元件直接相关。在调控的工作模型中，新生的AAP通过采用与Arg一起干扰核糖体a位点解码或其他对翻译至关重要的步骤的构象来阻止核糖体。拟议的研究将测试和完善aap介导的调节模型。具体目标是：1.；通过(a)确定停止的核糖体中是否含有与tRNA共价连接的新生AAP来确定肽基转移酶活性是否被阻断，(b)利用新生链与核糖体的光交联来检测新生AAP与核糖体在Arg作用下相互作用的构象变化。(c)利用Arg类似物的光交联来确定Arg是否与AAP或翻译机制相互作用，以及(d)确定调节是否只需要原核生物和真核生物之间保守的翻译成分。2. 检查无义介导的mRNA衰变（NMD）突变对mRNA稳定性和aap终止密码子识别的影响。NMD突变在体内消除了uorf编码的AAP的调节。3. 通过检测真菌中影响体内arg特异性调节或翻译的突变，确定对翻译控制重要的其他反式作用基因，以了解其对体外aap介导的核糖体停滞的影响。基因的克隆、鉴定和功能分析将为突变提供合理的解释。4. 通过对失速位点的突变分析，结合影响翻译的抗生素和影响调控的反式突变体的使用，深入了解调控功能。

DESCRIPTION (provided by applicant): The goal of this work is to understand how a peptide encoded by an upstream open reading frame (uORF) controls the movement of ribosomes on mRNA and regulates gene expression. The arginine attenuator peptide (AAP) is encoded by a uORF in mRNAs specifying a fungal arginine (Arg) biosynthetic enzyme and reduces gene expression when Arg is high. AAP-mediated regulation is observed in vitro using extracts from fungi and wheat germ. The nascent AAP causes the ribosome to stall in response to Arg. Stalled ribosomes block access to the downstream start coclon that initiates enzyme synthesis. The AAP is a unique eukaryotic peptide because its synthesis can arrest ribosomes involved in both termination and elongation. Its study provides exceptional opportunities to examine central translational processes that are still not understood. It will provide valuable information on events within the ribosome for evaluating the functions of antibiotics that interfere with translation. Since uORFs that control gene expression are found in animals, plants, fungi, viruses and bacteria, the proposed work is directly relevant to understanding these important regulatory elements.In a working model for regulation, the nascent AAP stalls the ribosome by adopting a conformation that, with Arg, interferes with decoding at the ribosomal A site, or with other steps crucial for translation. The proposed studies will test and refine models for AAP-mediated regulation. Specific aims are: 1. Directly examine the regulatory mechanism by (a) establishing whether stalled ribosomes contain the nascent AAP covalently linked to tRNA to determine whether peptidyl transferase activity is blocked, (b) using photocross-linking of the nascent chain to the ribosome to examine conformational changes in the interaction between the nascent AAP and the ribosome in response to Arg, (c) using photocros s-linking of Arg analogs to determine whether Arg interacts with the AAP or with the translational machinery, and (d) determining whether regulation requires only translational components conserved between prokaryotes and eukaryotes. 2. Examine mutations in nonsense-mediated mRNA decay (NMD) for their effects on mRNA stability and AAP-termination codon recognition. NMD mutations eliminate regulation by the uORF-encoded AAP in vivo. 3. Identify additional trans-acting genes important for translational control by examining mutations in fungi that affect Arg-specific regulation or translation in vivo for their effects on AAP-mediated ribosome stalling in vitro. Cloning and identification of the genes and analysis of their functions should provide a rational explanation of the mutations. 4. Obtain an in-depth understanding of regulatory function by mutational analysis of stalling sites in combination with the use of antibiotics affecting translation and trans-acting mutants affecting regulation.