The Mechanism by which Ribosomes Form Proteins

Hardesty 9315034将研究新生蛋白质在核糖体上线性合成时折叠成三维构象。 核糖体和分子伴侣在折叠过程中的作用将被评估。 三种酶（细菌二氢叶酸还原酶、氯霉素乙酰转移酶和哺乳动物罗丹酸酶）将在无细胞系统中通过与来自大肠杆菌的核糖体、tRNA和酶的偶联转录/翻译来合成。 通过荧光技术监测新生肽在核糖体上的肽基-tRNA上连续C-末端添加氨基酸而伸长时的折叠。 酶活性的获得将用作正确折叠成天然构象的量度。 对于涉及荧光的研究，将荧光团如香豆素掺入来自相应的氨酰基-tRNA衍生物如N（香豆素）-Mer-tRNA的新生肽中的特定位点，用于将香豆素掺入新生蛋白的N-末端。 将特别强调测试的假设，并确定这一折叠的哪一部分，如果有的话，发生在隧道或腔中的大亚基的核糖体。 分子伴侣在这三种酶的折叠中的作用也将被研究。 详细了解线性肽折叠成天然状态下蛋白质的特定三维构象所涉及的分子力和过程是目前分子生物学中最基本和最具挑战性的问题之一。 这个过程对于活性可溶性酶的形成至关重要，而不是像煮熟的白色那样，新形成的蛋白质聚集成不溶性的、难处理的变性蛋白质团。 了解这一过程对于合理设计和合成新酶至关重要。 理解折叠过程将是发展理论和程序的一大步，这将使人们有可能为研究，医学和工业生产新的蛋白质催化剂，其中许多可能不会发生在活细胞中。 但由于其固有的问题和技术上的困难，大多数研究都集中在蛋白质的变性复性上。我们的方法是根本不同的。 蛋白质在核糖体上通过从其N-末端到C-末端顺序添加氨基酸而线性合成。 我们使用荧光技术来监测新生蛋白质形成时的折叠。 已经开发了一种技术，通过该技术，我们将荧光团共价连接到特定的氨基酸作为氨酰-tRNA，然后将衍生的氨基酸掺入新生蛋白质中的独特点，因为它在核糖体上形成。 来自荧光团的荧光用于监测新生蛋白质的合成和折叠。 将研究核糖体本身和“伴侣”蛋白在折叠过程中的作用。 ***

Hardesty 9315034 Folding of nascent proteins into a three-dimensional conformation as they are synthesized linearly on ribosomes will be studied. The role of the ribosome and the chaperones in the folding process will be evaluated. Three enzymes (bacterial dihydrofolate reductase, chloramphenicol acetyl transferase, and mammalian rhodanese) will be synthesized by coupled transcription/translation with ribosomes, tRNA and enzymes derived from Escherichia coli in a cell-free system. Folding of the nascent peptides as they are elongated by the sequential, C-terminal addition of amino acids to peptidyl-tRNA on ribosomes will be monitored by florescence techniques. Acquisition of enzymatic activity will be used as a measure of correct folding into the native conformation. For studies involving florescence, a fluorophore such as coumarin will be incorporated at a specific site in the nascent peptide from the corresponding amino acyl-tRNA derivative, such as N(coumarin)-Mer- tRNA for incorporation of coumarin at the N-terminus of the nascent protein. Special emphasis will be given to testing the hypothesis, and to a determination of what part of this folding, if any, takes place within a tunnel or cavity in the large ribosomal subunit. The role of chaperones in folding of the three enzymes also will be studied. %%% A detailed understanding of the molecular forces and processes that are involved in folding of a linear peptide into the specific, three dimensional conformations of a protein in its native state is one of the most fundamental and challenging problems currently under intensive investigation in molecular biology. This process is crucial for the formation of active, soluble enzymes rather than aggregation of newly formed proteins into insoluble, intractable mass of denatured protein as occurs with egg white when it is cooked. An understanding of the process is critical for the rational design and synthesis of new enzymes. Understanding of the folding process would be a large step toward developing the theory and procedure that will make it possible to produce new protein catalysis for research, medicine and industry, many of which may not occur in living cells. Largely because of the inherent problems and technical difficulties, most studies have focused on refolding of proteins from their denatured state. Our approach is fundamentally different. Proteins are synthesized linearly on ribosomes by the sequential addition of amino acids from their N- terminal to C-terminal ends. We use florescence techniques to monitor folding of nascent proteins as they are formed. Techniques have been developed by which we covalently attached a fluorophore to a specific amino acid as aminoacyl-tRNA then incorporated the derivatized amino acid at a unique point in a nascent protein as it is formed on a ribosome. Fluorescence from the fluorophore is used to monitor synthesis and folding of the nascent protein. The role of the ribosome itself and "chaperone" proteins in the folding process will be studied. ***