Molecular determinants of A-site mRNA cleavage during ribosome pausing

描述（由申请人提供）：从细菌到人类的所有细胞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，在对应于核糖体A位点的位置切割信使RNA（mRNA）。这种A位点切割活性使mRNA不稳定，并可能代表一种新的基因调控机制。具体的假设是，新合成的新生蛋白质引发核糖体阻滞，其诱导核糖体催化A位点mRNA切割。该假说基于以下观察结果：1）A位点mRNA切割需要特定的新生肽序列，2）核糖体和翻译暂停是mRNA切割所需的，3）核糖体通常保护A位点mRNA免受核糖核酸酶的影响。基于这些观察，本建议的实验重点是确定A位点mRNA切割的分子要求。具体目标是：1.确定A位点mRNA切割的底物要求。将筛选支持有效切割的新生肽序列，并确定A位点密码子序列特异性。2.识别对A位点mRNA切割重要的核糖体组分。核糖体RNA对于解码A位点mRNA至关重要。假设核糖体RNA在翻译暂停事件期间A位点密码子的切割中起重要作用。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具有改变的A位点切割特性的核糖体变体。3.体外重建A位点mRNA切割反应。如果核糖体催化A位点切割，那么应该可以在由高度纯化的组分组成的限定的体外翻译系统中重建A位点切割反应。如果核糖体不催化A位点mRNA切割，则将在生物化学方法中使用定义的翻译系统来鉴定反式作用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.