Molecular determinants of A-site mRNA cleavage during ribosome pausing

描述（由申请人提供）：所有细胞，从细菌到人类，都使用核糖体暂停作为调节蛋白质表达的一般策略。在控制细胞生长和预防癌症的重要蛋白质合成过程中，受调节的核糖体暂停似乎特别常见。长期目标是了解一些暂停的核糖体如何引发独特的核糖核酸酶活性，将信使RNA （mRNA）切割到与核糖体a位点对应的位置。这种a位点的裂解活性破坏了mrna的稳定性，可能代表了一种新的基因调控机制。具体的假设是，新合成的新生蛋白引起核糖体阻滞，核糖体阻滞诱导核糖体催化a位点mRNA裂解。该假设基于以下观察：1)A-site mRNA的切割需要特定的新生肽序列；2)mRNA的切割需要核糖体和翻译停顿；3)核糖体通常保护A-site mRNA免受核糖核酸酶的攻击。基于这些观察结果，本课题的实验重点是确定a位点mRNA切割的分子要求。具体目标是：1。确定a位点mRNA切割所需的底物。新生肽序列将筛选支持有效切割的肽序列，并确定a位点密码子序列的特异性。2. 鉴定对a位点mRNA切割有重要作用的核糖体成分。核糖体RNA对解码a位点mRNA至关重要。假设核糖体RNA在翻译暂停事件中对A位点密码子的切割起重要作用。已知的突变和抗生素调节核糖体解码的保真度将研究对a位点mRNA切割的影响。此外，定向和无偏诱变方法将用于产生改变a位点切割特性的核糖体变体。3. 体外重建a位点mRNA裂解反应。如果核糖体催化了a位点的裂解，那么在由高度纯化的组分组成的定义的体外翻译系统中应该可以重建a位点的裂解反应。如果核糖体不催化a位点mRNA的裂解，则定义的翻译系统将用于生化方法来识别反式作用的a位点核酸酶。

DESCRIPTION (provided by applicant): All cells, from bacterial to human, use ribosome pausing as a general strategy to regulate protein expression. Regulated ribosome pausing appears to be particularly common during the synthesis of proteins that are important for controlling cell growth and preventing cancer. The long-term goal is to understand how some paused ribosomes elicit a unique ribonuclease activity that cleaves messenger RNA (mRNA) in a position corresponding to the ribosome A-site. This A-site cleavage activity destabilizes mRNAs and may represent a novel mechanism of gene regulation. The specific hypothesis is that the newly-synthesized nascent protein elicits a ribosome arrest, which induces the ribosome to catalyze A-site mRNA cleavage. The hypothesis is based on the observations that 1) A-site mRNA cleavage requires specific nascent peptide sequences, 2) the ribosome and translational pausing are required for mRNA cleavage, and 3) the ribosome usually protects A-site mRNA from ribonucleases. Based on these observations, the experimental focus of this proposal is to determine the molecular requirements of A-site mRNA cleavage. The specific aims are to: 1. Determine the substrate requirements for A-site mRNA cleavage. Nascent peptide sequences will be screened for those that support efficient cleavage, and A-site codon sequence specificity will be determined. 2. Identify ribosomal components that are important for A-site mRNA cleavage. Ribosomal RNA is critical for decoding A-site mRNA. Ribosomal RNA is hypothesized to play an important role in the cleavage of the A- site codon during translational pause events. Known mutations and antibiotics that modulate the fidelity of ribosome decoding will be studied for effects on A-site mRNA cleavage. In addition, directed and unbiased mutagenic approaches will be used to produce ribosome variants that have altered A-site cleavage properties. 3. Reconstitute the A-site mRNA cleavage reaction in vitro. If the ribosome catalyzes A-site cleavage, then it should be possible to reconstitute the A-site cleavage reaction in a defined in vitro translation system comprised of highly-purified components. If the ribosome does not catalyze A-site mRNA cleavage, the defined translation system will be used in a biochemical approach to identify the trans-acting A-site nuclease.