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.
