5S RNA Transport

核糖体在蛋白质的合成中起着关键作用， 活细胞，无论是植物细胞，动物细胞， 细菌 在真核生物中，这些复杂的巨大大分子 由四种核糖体RNA分子组成， 80种不同的核糖体蛋白。 核糖体组装发生在 细胞的核仁，其中四个RRNAS中的三个是 合成了 核糖体的所有其他成分都是合成的 转移到其他地方并运送到核仁进行组装。 这 研究项目的重点是识别和表征 运输途径和调节机制，使5S 核糖体RNA到核仁组装成核糖体。 的 研究将使用克隆的5S RNA基因的突变体及其 相关的蛋白质，试图破坏正常的核糖体组装或 功能，以便辨别每个组件如何在 正常的核糖体组装。 突变的基因或其基因产物 (5S RNA或5S RNA结合蛋白）将被显微注射到 非洲爪蟾卵母细胞，基因产物将通过 细胞通过放射性同位素标记和/或特异性 针对蛋白质或核蛋白组分的抗体。 在 将用分离的核仁进行体外交换研究 和分离的核糖体，结果将在 通过显微注射实验。 %%% 蛋白质合成是所有活细胞的基本过程。 蛋白质合成的机器是核糖体，一种复合体 由许多单独的大分子成分组成的结构， 蛋白质和多核糖核酸（RNA）。 的合成 核糖体是一个复杂的过程，因为合成的 单个大分子组分存在于聚合物的各个部分中， 细胞，有时在不同的时间，但有必要 把所有不同的部件组装在一起。 的 核糖体的蛋白质成分本身是在 预先存在的核糖体，这发生在细胞质中。 的 核糖核酸是在细胞核中合成的，因为它们 核糖体基因的直接转录物。 四种RNA中的三种 分子在细胞核的特定区域合成 称为核仁，首先是一个很长的转录本， 加工成三个小块。 是到了核仁 所有其他组件都已装配，包括 第四个也是最小的RRNA分子，称为“5S”。“这种5S RNA是 在细胞核中合成，但不在核仁中合成。 之前 该实验室的结果显示，在爪蟾卵母细胞中， 5S RNA先于其他RRNA组分合成， 并且在5S RNA转录后，它被转运到 细胞质，连同其自身的转录因子（蛋白质 （注：TFIIIA），并存储在那里。 随后，它成为 从TFIIIA解离并与不同的蛋白质缔合， L5，这是一种核糖体蛋白; 5S RNA-L5复合物， 运输回细胞核，特别是现在的核仁， 在那里组装成新生的核糖体。 这 该项目将解决这一机制和监管 复杂的核质运输。 这项工作是 意义不仅在于理解核糖体的生物发生， 这本身就是生物学中一个非常重要的问题， 了解一般核质运输，作为一个 这种现象是许多真核细胞 调控

Ribosomes play a key role in the synthesis of proteins in all living cells, whether they be plant cells, animal cells, or bacteria. In eukaryotes, these complex giant macromolecules are composed of four kinds of ribosomal RNA molecules and approximately eighty different ribosomal proteins. Ribosome assembly occurs in the nucleolus of the cell, where three of the four RRNAS are synthesized. All other components of the ribosome are synthesized elsewhere and transported to the nucleolus for assembly. This research project is focused on identifying and characterizing the transport pathways and regulatory mechanisms that bring the 5S ribosomal RNA to the nucleolus for assembly into ribosomes. The research will use mutants of the cloned 5S RNA genes and their associated proteins to try to disrupt normal ribosome assembly or function in order to discern how each component might function in normal ribosome assembly. The mutated genes or their gene products (5S RNAs or 5S RNA-binding proteins) will be microinjected in Xenopus oocytes, and the gene products will be followed through the cell by way of the radioisotopic labelling and/or specific antibodies against the protein or nucleoprotein components. In vitro exchange studies will be carried out with isolated nucleoli and with isolated ribosomes, and the results will be verified in vivo through the microinjection experiments. %%% Protein synthesis is a fundamental process of all living cells. The machinery for protein synthesis is the ribosome, a complex structure consisting of many individual macromolecular components, both proteins and polyribonucleic acids (RNAs). The synthesis of ribosomes is a complex process, since the synthesis of the individual macromolecular components occurs in various parts of the cell, and sometimes at different times, yet it is necessary to bring all the different components together for assembly. The protein components of the ribosome are themselves synthesized on preexisting ribosomes, and this takes place in the cytoplasm. The ribonucleic acids are synthesized in the nucleus, since they are direct transcripts of the ribosomal genes. Three of the four RNA molecules are synthesized in a specialized region of the nucleus termed the nucleolus, first as a very long transcript which is processed into the three smaller pieces. It is to the nucleolus that all the other components are brought for assembly, including the fourth and smallest RRNA molecule, termed "5S." This 5S RNA is synthesized in the nucleus, but not in the nucleolus. Prior results from this laboratory have shown that, in Xenopus oocytes, the 5S RNA is synthesized in advance of the other RRNA components, and that after the 5S RNA is transcribed it is transported to the cytoplasm, together with its own transcription factor (a protein termed TFIIIA), and is stored there. Subsequently, it becomes dissociated from TFIIIA and associates with a different protein, L5, which is a ribosomal protein; the 5S RNA-L5 complex is then transported back to the nucleus, specifically now to the nucleolus, where it becomes assembled into the nascent ribosomes. This project will address the mechanism and regulation of this complicated nucleocytoplasmic trafficking. The work is of significance not only for understanding ribosomal biogenesis, which is itself a very important problem in biology, but also for understanding nucleocytoplasmic trafficking in general, which as a phenomenon is fundamental to much of eukaryotic cellular regulation.