Peptide-mediated Pausing of Translation

核糖体是在所有类型的活细胞中将遗传密码翻译成蛋白质的细胞器。核糖体有两个亚基，一个小亚基负责解码遗传信息，一个大亚基负责将氨基酸偶联成蛋白质。新合成的蛋白质通过核糖体亚基中的长而窄的通道离开核糖体。在某些情况下，特定蛋白质的生长被短时间或长时间中断。该项目解决的机制，控制哪些蛋白质受到这种合成的阻止。目前的证据表明，停止涉及在聚合停止之前立即合成的蛋白质部分与隧道的RNA和/或蛋白质组分之间的相互作用。该项目的重点是一种非常短的蛋白质（CrbCmlA；只有9个氨基酸长），它参与调节另一种蛋白质（CmlA），使细胞对核糖体靶向抗生素氯霉素产生抗性。低浓度的氯霉素（不足以抑制核糖体合成其他蛋白质）需要阻止CrbCmlA的聚合，但目前尚不清楚氯霉素如何或为什么特异性地抑制这种蛋白质的合成。假设是生长中的蛋白质和氯霉素都与核糖体RNA和蛋白质相互作用，导致合成停止。本项目的实验旨在确定三种相互作用成分（CrbCmlA、核糖体和氯霉素）的哪些特征是这种三组分相互作用所必需的，以及这些相互作用如何建立翻译阻滞。之所以选择大肠杆菌作为实验生物，是因为它易于遗传操作，而且对其核糖体进行生化分析的程序也很容易获得。具体而言，该研究将采用遗传、生化和生理学的混合方法来(1)确定CrbCmlA暂停肽的关键特征（长度、序列和静电特性）、核糖体和暂停所需的共诱导剂，(2)表征CrbCmlA、核糖体成分和氯霉素之间的相互作用，以及(3)探测CrbCmlA对氯霉素与核糖体相互作用的影响。作为上下文，Crb的暂停将与另一种蛋白质（SecM）的翻译停止进行比较，后者不需要将单独的分子结合到核糖体上。这些实验的集体结果将为翻译阻滞的分子机制提供模型，并将两种蛋白质的实验结果进行比较，将揭示肽合成暂停是否可以用单一机制解释，还是核糖体中存在不同的暂停途径。这项研究将在两个方面产生影响。首先，它将对核糖体的功能产生更全面的了解，核糖体是所有类型生物细胞生存和生长所普遍需要的细胞器。其次，它将为研究生和本科生提供生物学研究方面的培训机会，将分子遗传学与主要多组分复合体中相互作用的高级生化探测结合起来。

The ribosome is the organelle that translates the genetic code into proteins in all types of living cells. The ribosome has two subunits, a small subunit that is responsible for decoding the genetic information, and a large subunit where coupling of amino acids into a protein takes place. Newly synthesized proteins exit the ribosome through a long, narrow tunnel in the large ribosomal subunit. Under some circumstances, growth of specific proteins is interrupted for short or long periods of time. This project addresses the mechanisms that control which proteins are subject to such arrest of synthesis. Current evidence indicates that the stoppage involves interactions between the portion of the protein that was synthesized immediately before the polymerization arrest and RNA and/or protein components of the tunnel. The project focuses on a very short protein (CrbCmlA; only 9 amino acids long), which is involved in the regulation of another protein (CmlA) that makes cells resistant to the ribosome-targeting antibiotic chloramphenicol. A low concentration of chloramphenicol (insufficient to inhibit ribosomes synthesizing other proteins) is required to arrest the polymerization of CrbCmlA, but it is not known how or why chloramphenicol specifically inhibits synthesis of this protein. The hypothesis is that the growing protein and chloramphenicol both interact with ribosomal RNA and proteins to bring about the conditions that stop synthesis. The experiments in this project are designed to define which features of each of the three interacting components (CrbCmlA, the ribosome and chloramphenicol) are required for this tri-component interaction and how these interactions establish translation arrest. The bacterium Escherichia coli is chosen as the experimental organism because of the ease of its genetic manipulation and the availability of procedures for biochemical analysis of its ribosomes. Specifically, the study will employ a mixture of genetic, biochemical, and physiological methods to (1) identify critical features of the CrbCmlA pausing peptide (length, sequence, and electrostatic properties), the ribosome, and the co-inducer necessary for pausing, (2) characterize the interactions between CrbCmlA, ribosomal components, and chloramphenicol, and (3) probe the effect(s) of CrbCmlA on the interaction of chloramphenicol with the ribosome. For context, pausing of Crb will be compared with translational arrest of another protein (SecM), which does not require binding of a separate molecule to the ribosome. The collective results of these experiments will provide model(s) for the molecular mechanism of translation arrest and comparison of the results for experiments with the two proteins will reveal whether peptide synthesis pausing can be explained by a single mechanism or if there are different pausing pathways in the ribosome.This research will have impact in two areas. First, it will generate a more comprehensive understanding of the function of the ribosome, an organelle that is universally required for the survival and growth of all types of biological cells. Second, it will provide training opportunities for both graduate and undergraduate students in biological research that integrate molecular genetics with advanced biochemical probing of interactions in a major multi-component complex.