Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective

Abstract

The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure. It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. The agreement between the higher-order structure of the small- subunit rRNA and protection against chemical modification is not perfect, however; some bases shown to covary canonically are accessible to chemical modification. For example, in both the small subunit and the isolated rRNA therefrom, position 66 is readily modified chemically; however, this position shows strong canonical covariation with position 103-there are over 30 phylogenetically independent examples among the bacteria, without exception. These discrepancies cannot be unequivocally interpreted. They could reflect the 16S rRNA or the 30S subunit not being in a functionally optimal state in vitro; they might imply that the bases inferred as paired by comparative analysis are not paired throughout the entire translation cycle. However, it is highly unlikely that discrepancies of this kind imply that these bases (at positions 66 and 103) do not pair at all. It is interesting that the protections that hold for the isolated rRNA do not agree with those shown by the corresponding intact subunit in a few cases, which could be interpreted to mean that the functional structure of rRNA, although primarily inherent in the rRNA itself, is secondarily determined by association with protein. Along these lines, we would like to make a more general point. Comparative analysis has played a very strong role in determining the structure of several RNA molecules, including a smaller, highly variable RNA, the RNA moiety of RNase P. In this case many of the predictions have been tested and a 'minimal' functional form of the molecule has been predicted, genetically engineered, and shown to be functional. At this junction we question what additional information about RNA structure can be inferred by using comparative methods. Our comparative rationale for RNA structure determination is based on the simple concept of a homologous structure for the RNA molecule under study. Our primary method for identifying this isomorphic structure relies on the search for compensatory base substitutions or positional covariance, which has revealed a secondary structure and the beginnings of its tertiary structure for the 16S and 23S rRNAs. This search has, to a first approximation, identified the structural elements in common with all sequences in their respective data sets. Further refinements in rRNA structure will also come from a more exhaustive and quantitative analysis of the 16S and 23S rRNA data sets. Newer quantitative correlation algorithms, under development, are more sensitive than previous methods and are beginning to identify helical base pairings constrained by surrounding base pairs and other nucleotides not considered to be directly involved in structural interactions. With the ever-increasing 16S and 23S rRNA sequence collections and the more powerful and dynamic correlation analysis algorithms, we should expect to find more structural constraints in these RNA molecules, many of which will not involve positions that covary in the strict one-to-one manner. Ultimately, comparative methods will go beyond the search for positions that covary in a simple one-to-one manner as the paradigm for homologous structure. Instead, these methods will utilize a growing appreciation for RNA conformations and a mapping between sequence and its secondary and tertiary structure to assist in the search for isomorphic structure. The comparative method, although inferring base pairings, does not imply or require that all of these pairings occur simultaneously. Unfortunately, the manner in which these secondary-structure figures are drawn suggests just this, a static structure devoid of possible structural alternations. The essence of ribosomal function most probably involves dynamic movement in rRNA structure. Transforming these static 16S and 23S rRNA structures presented here into a functioning ribosome will entail experimental approaches. The ultimate goal of all analysis of the ribosome is to determine its structure and relate this to its function. Comparative analysis clearly cannot carry rRNA structure to this point. Only a combination of approaches can produce the picture of the functioning ribosome.