Nucleic Acids Research

We have derived a secondary structure model for 16S ribosomal RNA on the basis of comparative sequence analysis, chemical modification studies and nuclease susceptibility data. Nucleotlde sequences of the E_. col 1 and B. brevls 16S rRNA chains, and of RNAse Tj oligomer catalogs from 16S rRNAs of over 100 species of eubacteria were used for phylogenetic comparison. Chemical modification of G by glyoxal, A by m-chloroperbenzolc acid and C by bisulfite in naked 16S rRNA, and G by kethoxal In active and Inactive 30S ribosomal subunits was taken as an indication of single stranded structure. Further support for the structure was obtained from susceptibility to RNases A and Ti . These three approaches are in excellent agreement. The structure contains fifty helical elements organized into four major domains, in which k(> percent of the nucleotides of 16S rRNA are Involved in base pairing. Phylogenetic comparison shows that highly conserved sequences are found principally in unpaired regions of the molecule. No knots are created by the structure. INTRODUCTION Our understanding of the mechanics of translation today is all but rudimentary, despite two decades of Intensive work on the subject. This may reflect the complexity of the mechanism. Yet, it may Just as well reflect misplaced emphases. There has been a strong tendency to picture the ribosome function as somehow defined by its protein components, which Is partly responsible for the heavy emphasis placed upon characterization of ribosomal proteins, protein factors, etc. Ribosomal RNAs, on the other hand, have tended to be viewed as "structural," as providing a sort of scaffolding upon which to position the function-defining proteins. However, the fact (revealed first by nucleic acid hybridization [1]) that ribosomal RNA sequence is highly conserved phylogenetleally suggests these molecules to be somewhat more than mere scaffolding. This has been borne out by a variety of studies, which provide evidence for the direct participation of 16S ribosomal RNA In messenger RNA selection [2-k], tRNA binding [5"7], ribosomal subunit association [8-10], and antibiotic sensitivity/resistance [llj. © IRL Prow Limited, 1 Falconbsrg Court, London W 1 V BFG. U.K. 2275 Nucleic Acids Research In any case, this matter will soon be resolved. The capacity to determine nucleic acid sequences easily Is now at hand; for this reason alone a number of rlbosomal RNAs will ultimately be sequenced. The availability of the sequences of 16S and 23S rRNA from E_. col I [ 12— 1 **] has made Is possible to begin attacking the problem of rlbosomal RNA secondary structure. Experience with the small functional RNAs, 5SrRNA and tRNA, give an Indication of the problems that will be encountered in determining secondary structure for the much larger ribosomal RNAs. In both of the former cases the secondary structure of the molecule was not established until comparative sequence data was employed [15, 16]. For the larger rlbosomal RNAs, which contain a bewildering array of possible double stranded structures, the difficulties in sorting out the true secondary structural elements are far greater. As we shall see, It is unsafe to make the restrictive energetic assumptions that would reduce drastically the number of possible structures; In so doing one can eliminate some of the true helices. Furthermore, one is not justified at this stage In assuming that two different helices whose sequences overlap are mutually exclusive; It is possible that both exist but at different times in the ribosomal cycle. It may even be unsafe to assume— as our computer search does—that double helical structure Is determined solely by the normal Watson-Crick base pairs and G-U pairs. The limitations of a comparative approach to secondary structure are obvious: Only secondary structural elements that are themselves conserved will be detected by the method. In a strict sense the approach demonstrates only that a pairing constraint exists, not that actual physical pairing occurs. Nevertheless, it Is unlikely that actual pairing does not occur In most If not all of these instances. In practical terms, comparative evidence is not sufficient to define the extent of a double helical element; It merely reveals Its existence. Because of this and the possibilities that non-WatsonCrlck pairs are involved In helical structure and that not all helices are present all of the time, It Is useful to provide additional evidence for the actual physical presence of the helical structures In rRNA. Therefore, we have also studied the relative reactivity of selected residues In the rRNA to various chemical modifying reagents that respect secondary structure, and have catalyzed the susceptibility of various residues to nuclease attack. Once comparative evidence has established the existence of a helical element, these criteria can then define Its extent, and so on. The present study Is confined to the I6S rRNA from the true bacteria (eubacterla). The comparative data base includes the E_. col i sequence [12,13],