Mechanisms of RNA processing and decay that are dependent on RNaseE and related enzymes

依赖于 RNaseE 和相关酶的 RNA 加工和降解机制

• 批准号: BB/D016096/1
• 负责人: Kenneth McDowall
• 金额: $ 35.46万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD016096%2F1
• 关键词: Mechanisms; RNA; processing; decay; dependent

In response to changing and often adverse or competitive environments, organisms fine-tune their metabolism and composition via changes in the expression of genes (the basic units of information inherited in all organisms) in order to maximize survival and growth. An important model for studying gene expression is the gut-dwelling bacterium E. coli. This is due in part to the ease with which this organism can be manipulated genetically and grown using simple media. It was experiments with E. coli that finally proved that DNA encodes the inheritable information (Hershey & Chase), deciphered the relationship between the information in genes and the amino acid building blocks of proteins (Nirenberg & Khorana), which form most of the working parts of cells, and demonstrated that E. coli can sense the availability of nutrients and then adjust its metabolism to use first those that provide the most 'energy' (Jacob & Monod). It was found, as part of work on the latter, that the synthesis of proteins required for the utilisation of a sugar was terminated rapidly when the sugar was no longer available. This led to the suggestion, which has now been proved, that protein is encoded via an unstable messenger that rapidly disappears when its synthesis is blocked. This intermediate is now known to be composed of RNA and is in effect a 'copy' of the information in genes. The process of making messenger RNA from DNA (transcription) and the process of making proteins from mRNA (translation) have been studied extensively using biochemistry and genetics. More recently, the solving of atomic-resolution structures of the machines that mediate transcription and translation has provided paths to an understanding of the molecular mechanisms at the heart of these steps in gene expression. Although the synthesis of mRNA and its subsequent translation are clearly important steps, the rate of decay of any RNA is just as important as its rate of synthesis in determining the cellular levels. Consequently, the stability of mRNA is a key determinant of the amount of protein produced from a gene. Despite the central importance of mRNA decay, our understanding of this process lags behind that of transcription and translation. Excellent progress is however being made. A major breakthrough in the study of mRNA decay in E. coli was the identification of an essential gene that is required for the normal rapid decay of many mRNAs. This gene has been shown not only to encode an endoribonucleolytic activity (RNaseE), but also to serve as a platform for the assembly of a machine called the degradosome, which contains other enzymes important for rapid mRNA decay. To better understand the process of mRNA decay in E. coli, we have investigated the factors that control the cleavage of RNA by RNaseE. This has involved using chemistry to synthesize substrates that can be used in biochemical assays and genetics to knockout or modify gene function in vivo. Biophysical techniques have been used to establish the overall structure of the catalytic domain of RNaseE and features that are required for both its assembly and ribonucleolytic activity. Most recently, one of our collaborations has led to the solving of the crystal structure of the catalytic domain of E. coli RNaseE. Using the considerable detail this has provided at the atomic level, our overall objective now is to establish the contribution of specific molecular traits of RNaseE (and associated proteins) to the pattern of RNA processing and decay observed in E. coli. This in turn may permit more efficient use of E. coli as a host for producing biomolecules of commercial or medical importance and could eventually be useful in the development of antibacterial drugs that target mRNA decay mechanisms.

为了应对不断变化的，往往是不利的或竞争性的环境，生物体通过改变基因（所有生物体中遗传的基本信息单位）的表达来微调其代谢和组成，以最大限度地提高生存和生长。研究基因表达的一个重要模型是肠道细菌E。杆菌这部分是由于这种生物可以很容易地进行遗传操作和使用简单的培养基生长。用E.大肠杆菌最终证明了DNA编码的遗传信息（Hershey & Chase），破译了基因中的信息和蛋白质的氨基酸构建块之间的关系（Nirenberg & Khorana），蛋白质构成了细胞的大部分工作部分，并证明了大肠杆菌。大肠杆菌可以感觉到营养物质的可用性，然后调整其新陈代谢，首先使用那些提供最多“能量”的营养物质（Jacob & Monod）。作为对后者的研究的一部分，人们发现，当糖不再可用时，利用糖所需的蛋白质合成迅速终止。这导致了现在已经被证明的建议，即蛋白质是通过一种不稳定的信使编码的，当其合成受阻时，这种信使会迅速消失。现在已知这种中间体由RNA组成，实际上是基因中信息的“副本”。利用生物化学和遗传学对从DNA制造信使RNA（转录）和从mRNA制造蛋白质（翻译）的过程进行了广泛的研究。最近，对介导转录和翻译的机器的原子分辨率结构的解决为理解基因表达中这些步骤的核心分子机制提供了途径。虽然mRNA的合成及其随后的翻译显然是重要的步骤，但在决定细胞水平方面，任何RNA的衰变速率与其合成速率一样重要。因此，mRNA的稳定性是基因产生蛋白质量的关键决定因素。尽管mRNA衰变至关重要，但我们对这一过程的理解落后于转录和翻译。不过，目前正在取得很大进展。在E.在大肠杆菌中，鉴定了许多mRNA正常快速衰变所需的一个必需基因。该基因已被证明不仅编码核糖核酸内切酶（RNaseE）活性，而且还作为一个平台，用于组装一个称为降解体的机器，其中包含其他对快速mRNA降解很重要的酶。为了更好地了解E.在大肠杆菌中，我们研究了RNaseE切割RNA的控制因素。这涉及使用化学合成可用于生物化学测定和遗传学的底物，以在体内敲除或修饰基因功能。生物物理技术已被用来建立RNaseE的催化结构域的整体结构和所需的装配和核糖核酸裂解活性的功能。最近，我们的一项合作已经解决了E. coliRNaseE.利用这在原子水平上提供的大量细节，我们现在的总体目标是建立RNaseE（和相关蛋白质）的特定分子特征对在E.杆菌这反过来又可以允许更有效地使用E。大肠杆菌作为生产具有商业或医学重要性的生物分子的宿主，并最终可用于开发靶向mRNA衰变机制的抗菌药物。