Control of arg-2 Gene Expression in Neurospora

描述(申请人提供)：这项工作的目标是了解由上游开放阅读框架(UORF)编码的多肽如何控制核糖体在mRNA上的运动和调节基因表达。精氨酸衰减器肽(AAP)是由mRNAs中指定真菌精氨酸(Arg)生物合成酶的uORF编码的，当Arg含量较高时，AAP会降低基因表达。使用真菌和小麦胚芽的提取物，在体外观察到AAP介导的调节。新生的AAP导致核糖体对Arg的反应停滞。停滞不前的核糖体阻止了启动酶合成的下游起始密码子的访问。AAP是一种独特的真核多肽，因为它的合成可以阻止参与终止和延伸的核糖体。它的研究为检验仍未被理解的中心翻译过程提供了难得的机会。它将为评估干扰翻译的抗生素的功能提供关于核糖体内事件的有价值的信息。由于在动物、植物、真菌、病毒和细菌中都发现了控制基因表达的uORF，因此这项拟议的工作与理解这些重要的调控元件直接相关。在一个调控的工作模型中，新生的AAP通过采用一种构象来拖延核糖体，该构象与Arg一起干扰核糖体A位点的解码或其他对翻译至关重要的步骤。拟议的研究将测试和完善AAP介导的调控模型。具体目的是：1.直接研究调控机制，方法是：(A)确定停滞的核糖体是否含有与tRNA共价连接的新生AAP，以确定多肽转移酶活性是否被阻断；(B)利用新生核糖体与核糖体之间的光交联链来检测新生AAP与核糖体之间相互作用的构象变化，以响应Arg；(C)利用Arg类似物的光Cros S连接来确定Arg是与AAP相互作用还是与翻译机制相互作用；以及(D)确定调控是否只需要原核生物和真核细胞之间保守的翻译成分。2.检测无义介导的信使核糖核酸衰变(NMD)中突变对信使核糖核酸稳定性和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.通过检测真菌中影响体内Arg特异性调控或翻译的突变，以确定它们对AAP介导的体外核糖体停滞的影响，从而确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对其功能的分析应该为突变提供合理的解释。4.结合使用影响翻译的抗生素和影响调控的反式作用突变体，通过对停滞位点的突变分析，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.