RIBOSOMAL RNA ANALOGS AND THE MECHANISMS OF TRANSLATION

也许令人惊讶的是， 负责mRNA翻译的蛋白质在很大程度上仍然没有被表征， 超过30年的调查。这些问题的棘手性 可能是由于规模大和复杂的 翻译器，特别是核糖体。即使是小的（30 S） E.例如，大肠杆菌核糖体含有一个拷贝的16 S rRNA（1542个核苷酸）和21种不同的蛋白质，产生一种聚集体 分子量为930，000道尔顿。显然， 需要研究翻译机制。一个重要的和统一的 在过去的十年里出现的一种观点是，核糖体RNA是 与核糖体功能的几乎所有方面都密切相关。 受这一假设以及RNA领域其他最新进展的刺激， 生物化学，我们已经开始研究功能性 潜在的16 S rRNA的一个小子域，解码区，在 小的无蛋白质RNA构建体或寡核苷酸类似物的形式。 这样的寡核苷酸类似物可能极大地简化分析 参与翻译的分子机制，因为它们 结构相对简单。与联合国的活动相一致 在核糖体的解码区，我们已经发现， 可以参与功能上重要的 与抗生素、mRNA和tRNA的相互作用， 剩余的16 S rRNA的1500个核苷酸和所有的核糖体蛋白。 这些观察结果构成了第一次证明的功能 在完全不存在蛋白质的情况下，核糖体RNA的活性，并建议 强烈地，基本翻译机制，如解码， 基于RNA的。该提案的目标是实现这一潜力， 新的方法，最初通过扩展我们的初步研究，测试 假设，首先，除了那些已经牵连，其他 mRNA和tRNA配体中的原子，特别是骨架原子， 参与这些相互作用，第二，密码子--反密码子 相互作用，即解码，可以由类似物介导。三是 将利用这种新方法将强大的体外遗传 选择技术的解码区域的第一次。这些 研究将构成人工系统发育分析，结果 可以直接与16 S的超大数据库进行比较， rRNA序列。第四，我们会展开合作研究， 通过X确定解码区域的三维结构， 射线晶体学，应用最近发展和非常成功 测量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亚结构域 核糖体内; 23 S rRNA的肽基转移酶区域。目标 这些研究的最终目的是表明，肽基转移酶 在完全不存在蛋白质的情况下由23 S rRNA催化，同时还 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.