Third International Symposium on Catalytic RNAs (Ribozymes) and Targeted Gene Therapy of the Treatment of HIV Infection, San Diego, CA 1992

Comparative Analysis of the Higher-order Structure of Ribonuclease P RNA.

James W. Brown, Elizabeth S. Haas, and Norman R. Pace, Department of Biology and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, IN 47405

Ribonuclease P (RNase P) cleaves leader sequences from precursors of tRNA to generate the mature 5' end of the tRNA. Bacterial RNase P occurs in vivo as a complex containing a small protein (119 amino acids in E. coli) and a much larger RNA (377 nucleotides). At high ionic strength, in vitro, the RNA alone is an efficient and accurate catalyst; it is the only known naturally-occuring RNA that functions as an enzyme, in the sense that each molecule of RNase P RNA processes many substrate molecules.

An accurate model for the structure of a RNA is an essential prerequiste to informative studies of its function. The most successful approach to inferring the secondary structures of RNAs has been the use of phylogenetic comparative analysis. Using this method, a model for the secondary structure of bacterial RNase P RNA has been developed and is currently being extended and refined to the base-pair level. The RNase P RNA gene sequences from representatives of 6 evolutionarily distant phylogenetic groups of Bacteria (27 sequences in all) have so far been determined. These genes are highly diverse in sequence (only 35 - 55% identical), yet the secondary structures of the encoded RNAs are strikingly similar. Most of the variation in RNase P RNA secondary structure is, except in the case of Bacillus spp., length-variation in a small number of hairpins in the molecule. The Bacillus structures are significantly altered with respect to the remaining RNase P RNAs; several large insertions and deletions have occurred in otherwise conservative regions of the molecule. Nevertheless, the conservative "core" of the structure remains in the Bacillus RNAs. The core structure has been shown to contain all of the elements essential for catalysis. Analysis of one of the variably-present elements suggests that the tertiary structures of the Bacillus and E. coli RNAs may more closely resemble one another than the secondary structures would imply.

The secondary structure model of bacterial RNase P RNA is becoming sufficiently well-defined that specific regions of the molecule can be modeled in three dimensions. The sequences from Thermotoga spp. provide particularly useful constraints, because they have shorter connections between helical elements than other RNase P RNAs; these minimal connections must be consistant with the conserved tertiary structure. The secondary structure model of RNase P RNA contains two pseudoknots, one of which is structurally analogous to the "H-type" pseudoknots of some plant viral RNAs, the other may contain a three-stranded helix comprised of the most highly conserved sequences in the RNA. A four-helix intersection with no intervening nucleotides elsewhere in the RNase P RNA secondary structure is likely an A-form analog of a Holliday junction. Variations in helix lengths (or presence) have been used to identify stacking interactions and the orientation of helices within the global structure of the RNA.