Control of arg-2 Gene Expression in Neurospora

脉孢菌中 arg-2 基因表达的控制

• 批准号: 6545478
• 负责人: MATTHEW Steven SACHS
• 金额: $ 31.52万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6545478
• 关键词: Neurospora; Saccharomyces cerevisiae; aminoacid biosynthesis; arginine; fungal genetics; gene expression; gene mutation; genetic mapping; genetic regulation; genetic regulatory element; genetic translation; iodine; messenger RNA; methionine; mutant; nucleic acid sequence; open reading frames; protein isoforms; protein structure function; radionuclides; ribosomes; site directed mutagenesis; sulfur; transfer RNA; translation factor

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.

描述（由申请人提供）：这项工作的目标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 mRNA 中的 uORF 编码，指定真菌精氨酸 (Arg) 生物合成酶，并在 Arg 高时降低基因表达。使用真菌和麦芽提取物在体外观察到 AAP 介导的调节。新生的 AAP 导致核糖体响应 Arg 而停滞。停滞的核糖体阻止进入启动酶合成的下游起始克隆。 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。它的研究提供了绝佳的机会来检查仍然不被理解的中心翻译过程。它将提供有关核糖体内事件的有价值的信息，以评估干扰翻译的抗生素的功能。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通过采用以下构象来阻止核糖体：与 Arg 一起干扰核糖体 A 位点的解码，或干扰对翻译至关重要的其他步骤。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. 通过以下方式直接检查调节机制：(a) 确定停滞核糖体是否含有与 tRNA 共价连接的新生 AAP，以确定肽基转移酶活性是否被阻断，(b) 使用新生链与核糖体的光交联检查新生 AAP 和核糖体之间相互作用中响应 Arg 的构象变化，(c) 使用光交叉 s-连接Arg 类似物以确定 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组件。 2. 检查无义介导的 mRNA 衰变 (NMD) 中的突变对 mRNA 稳定性和 AAP 终止密码子识别的影响。 NMD 突变消除了体内 uORF 编码的 AAP 的调节。 3. 通过检查影响 Arg 特异性调节或体内翻译的真菌突变及其对体外 AAP 介导的核糖体停滞的影响，鉴定对翻译控制重要的其他反式作用基因。基因的克隆和鉴定以及其功能的分析应该为突变提供合理的解释。 4. 通过对停滞位点的突变分析并结合影响翻译的抗生素和影响调节的反式作用突变体的使用，深入了解调节功能。