Functional Analysis of the Ribosome CAR Surface

项目总结/摘要 基因表达在多个水平上受到控制，这些水平是整合的，因此细胞、组织和生物体可以 快速有效地响应不断变化的环境。mRNA的调节转录之后是 整合翻译控制机制以确定蛋白质生产水平。我们提出的 研究的目的是调查一层翻译调控，这取决于一个新发现的 核糖体相互作用表面，其锚定到核糖体A位点解码中心，允许其与 紧接着进入A位点的+1密码子。这种“CAR”相互作用表面由三个保守的 16S/18S rRNA C1054、A1196（E. coli编号）和核糖体蛋白S3 R146（S.酿酒酵母 编号;在真核生物中保守）-并且在易位至tRNA核苷酸的开始处锚定 (nt)34，密码子摆动核苷酸的反密码子配偶体。CAR曲面具有有趣的性质 当+1密码子符合GCN序列时，其相互作用最强。从最初的密码- 一个基因ORF的斜坡区在决定核糖体被转录的速率方面特别有影响。 启动了mRNA，斜坡区域符合GCN密码子的程度可能决定了 每个基因对这一层调控的敏感性。 核糖体生物学中的一个新兴观点是，不同形式的核糖体是在不同的细胞环境下产生的。 条件，允许细胞根据其环境调节蛋白质翻译。我们的分析表明 新产生的核糖体是否在解码中心有不同的残基修饰， 邻居-R146，CAR残基之一，以及几个rRNA核苷酸-可以决定是否 CAR界面可以与+1密码子相互作用。这些修饰的酶被下调 我们的分子动力学分析表明，当这些修饰 目前，CAR与+1密码子的相互作用相当弱。我们将测试修改水平是否 在目标1的压力下减少。我们还计划研究在压力和压力下核糖体对mRNA的行为， 使用核糖体分析实验评估CAR和CAR之间的相互作用是否在非应激条件下进行。 +1 GCN密码子可能会影响翻译过程中核糖体沿着mRNA移动的速率（Aim 2）。由于将CAR锚定到tRNA反密码子的tRNA nt 34通常含有各种修饰， 根据压力，我们将进行分子动力学分析，以测试这些修饰是否发生变化 CAR锚定到反密码子，从而改变CAR与+1密码子的相互作用（目的3）。 该项目的重点是整合体内测试和核糖体动力学的计算建模， 成功的，将导致重要的见解到一个新的层次的翻译调控，并为学生提供 深入介绍了在分子水平上分析系统，以及跨学科的模式， 研究揭示了这个重要的生物系统的潜在行为。

PROJECT SUMMARY/ABSTRACT Gene expression is controlled at multiple levels that are integrated so that cells, tissues and organisms can respond efficiently and quickly to changing environments. Regulated transcription of mRNAs is followed by integrated mechanisms of translational control to determine levels of protein production. Our proposed research is aimed at investigating a layer of translational regulation that depends upon a newly-identified ribosome interaction surface that is anchored to the ribosome A-site decoding center, allowing it to H-bond with the +1 codon that is next to enter the A site. This “CAR” interaction surface consists of three conserved residues—16S/18S rRNA C1054, A1196 (E. coli numbering) and ribosomal protein S3 R146 (S. cerevisiae numbering; conserved in eukaryotes)—and is anchored at the beginning of translocation to tRNA nucleotide (nt) 34, the anticodon partner of the codon wobble nucleotide. The CAR surface has the interesting property that its interactions are strongest when the +1 codon conforms to a GCN sequence. Since the initial codons— the ramp region—of a gene’s ORF are particularly influential in determining the rate at which ribosomes are launched down the mRNA, the extent to which ramp regions conform to GCN codons likely determines the sensitivity of each gene to this layer of regulation. An emerging idea in ribosome biology is that different versions of ribosomes are made under various cellular conditions, allowing cells to regulate protein translation according to their environments. Our analysis suggests that whether newly made ribosomes have modifications of various residues in the decoding center neighborhood—R146, one of the CAR residues, as well as several rRNA nucleotides—may determine whether the CAR interface can interact with the +1 codon. The enzymes for these modifications are down-regulated under stress conditions, and our molecular dynamics analysis indicates that when these modifications are present, the interaction of CAR with the +1 codon is considerably weaker. We will test if modification levels are reduced under stress in Aim 1. We also plan to examine ribosome behavior on mRNAs under stressed and unstressed conditions using ribosome profiling experiments to assess whether interactions between CAR and the +1 GCN codons may influence the rates at which ribosomes travel along the mRNA during translation (Aim 2). Since tRNA nt 34, which anchors CAR to the tRNA anticodon, often contains various modifications that are stress-dependent, we will carry out molecular dynamics analysis to test whether these modifications change anchoring of CAR to the anticodon with consequent changes in interactions of CAR with the +1 codon (Aim 3). This project, with its focus on integration of in vivo testing and computational modeling of ribosome dynamics, if successful, will lead to important insights into a new layer of translational regulation, and provide students with in-depth introductions to analysis of a system at the molecular level, as well as interdisciplinary modes of inquiry that reveal underlying behaviors of this biologically important system.