RIBOSOMAL RNA ANALOGS AND THE MECHANISMS OF TRANSLATION

也许令人惊讶的是，特定的分子机制 负责翻译的信使核糖核酸在很大程度上仍未确定 经过30多年的调查。这些问题的棘手之处 很可能归因于 翻译机构，特别是核糖体。即使是小的(30多岁) 例如，大肠杆菌核糖体的亚基包含一个16S的拷贝 RRNA(1542个核苷酸)和21种不同的蛋白质，产生一个聚合 分子量为93万道尔顿。显然，新的方法 研究翻译机制是必要的。一个重要而统一的问题 过去十年出现的想法是，核糖体RNA是 密切参与核糖体功能的几乎所有方面。 受这一假说和RNA的其他最新发展的刺激 生物化学，我们已经开始了表征功能的研究 16S rRNA的一个小亚域，即解码区，在 一种无蛋白的小RNA结构形式，或寡核苷酸类似物。 这种寡核苷酸类似物有可能极大地简化分析 翻译中涉及的分子机制，因为它们的 相对结构简单性。与本组织的活动相一致 在核糖体的解码区，我们发现了一种寡核苷酸类似物 的译码区可以参与功能显著的 与抗生素、mRNA和tRNA的相互作用 其余1500个核苷酸的16S rRNA和所有核糖体蛋白。 这些观察构成了功能的第一次演示 在完全没有蛋白质的情况下核糖体RNA的活性，并提示 强烈地认为，基本的翻译机制，如解码，是 基于RNA的。这项提议的目标是实现这一潜力 新方法，最初是通过扩展我们的初步研究，测试 首先，假设除了那些已经牵连的假设之外，还有其他假设 原子，特别是mRNA和tRNA配体中的主干原子， 参与这些相互作用，第二，密码子--反密码子 交互，也就是解码，可以由类比来中介。第三，我们 将利用这一新方法将强大的体外基因 首次将选择技术引入到解码区。这些 研究将构成人工系统发育分析的结果 其中可以直接与16秒的超大数据库进行比较 RRNA序列。第四，我们将启动合作研究，旨在 通过X-确定解码区的三维结构 射线结晶学，应用最新发展并取得很大成功 测定RNA结晶条件的方法。最后，我们将 将我们的策略应用于其他功能相关的RNA亚域 在核糖体内；23S rRNA的肽基转移酶区域。目标是 这些研究表明，最终，肽基转移酶是 在完全没有蛋白质的情况下由23S rRNA催化，同时也 为今后的结构研究奠定了坚实的基础。

It is, perhaps, surprising that the specific molecular mechanisms responsible for translation of mRNA remain largely uncharacterized after more than 30 years of investigation. The intractability of these problems is probably attributable to the large size and complexity of the translational apparatus, specifically ribosomes. Even the small (30S) subunit of the E. coli ribosome, for example, contains one copy of 16S rRNA (1542 nucleotides) and 21 different proteins, producing an aggregate molecular weight of 930,000 daltons. Evidently, new approaches for studying translational mechanisms are needed. One important and unifying idea that has emerged over the past decade is that ribosomal RNA is intimately involved in virtually all aspects of ribosome function. Stimulated by this hypothesis, and by other recent developments in RNA biochemistry, we have initiated studies characterizing the functional potential of a small subdomain of 16S rRNA, the decoding region, in the form of a small protein-free RNA construct, or oligonucleotide analog. Such oligonucleotide analogs can potentially simplify enormously analysis of the molecular mechanisms involved in translation because of their comparative structural simplicity. Consistent with the activities of the decoding region in ribosomes, we have found that an oligonucleotide analog of the decoding region can participate in functionally significant interactions with antibiotics, mRNA, and tRNA in the absence of the remaining 1500 nucleotides of 16S rRNA and all of the ribosomal proteins. These observations constitute the first demonstration of the functional activity of ribosomal RNA in the complete absence of protein, and suggest strongly that fundamental translational mechanisms, like decoding, are RNA-based. The goals of this proposal are to fulfill the potential of this new approach, Initially by extending our preliminary studies, testing the hypotheses, first, that in addition to those already implicated, other atoms, particularly backbone atoms in the mRNA and tRNA ligands, participate in these interactions, and second, that codon--anticodon interaction, that is decoding, can be mediated by the analog. Third, we will exploit this new approach to apply powerful in vitro genetic selection techniques to the decoding region for the first time. These studies will constitute an artificial phylogenetic analysis, the results of which can be compared directly to the extremely large database of 16S rRNA sequences. Fourth, we will initiate collaborative studies aimed at determining the three-dimensional structure of the decoding region by X- ray crystallography, applying recently developed and highly successful methods for surveying RNA crystallization conditions. Lastly, we will apply our strategy to the other functionally implicated RNA subdomain within the ribosome; the peptidyl transferase region of 23S rRNA. The goal of these studies is to show, ultimately, that peptidyl transferase is catalyzed by 23S rRNA in the complete absence of proteins, while also laying a strong foundation for future structural studies.