Second Int. Meetings on Structure, Mechanism and Function of Ribonucleases, Sant Feliu de Guxols, Spai, 1990

Structure, function and evolution of ribonuclease P RNA.

James W. Brown*, Elizabeth S. Haas, Alex B. Burgin, Sylvia C. Darr, Dirk A Hunt, Drew Smith, and Norman R. Pace. Department of Biology, Indiana University, Bloomington, IN 47405 USA

Ribonuclease P cleaves leader sequences from pre-tRNAs to generate the mature 5' end of tRNA. In the eubacteria Bacillus subtilis and Escherichia coli, RNase P is composed of a small protein (119 amino acids) and a large RNA (~400 nucleotides). At high salt concentrations in vitro, the RNA alone is an efficient and accurate catalyst.

The secondary structures of the eubacterial RNase P RNAs are being elucidated using a phylogenetic comparative approach. Genes encoding RNase P RNA from each recognized sub-group of the purple "phylum" of eubacteria ("proteobacteria") have now been determined. These RNase P RNA sequences allow the refinement, to the base-pair level, of the phylogenetic model for RNase P RNA secondary structure. Additional evidence for all previously identified helices has been obtained; in some cases these helices have been lengthened by new covariation or shortened by non-covariation of potential base-pairings. Previously unobserved secondary and higher-order structural covariations, including conserved non-canonical pairings, have been identified. These additional sequences have also been used to construct a parsimonious model for the evolution of RNase P RNA primary and secondary structure in purple eubacteria and Bacillus, and allows the reconstruction of ancestral RNase P RNA structures. Evolutionary change among the RNase P RNAs occurs primarily in descrete structural domains that are peripheral to a highly conserved "core" structure. Analysis of evolutionary changes in the phylogenetic group-specific structural elements has been used to design and construct synthetic "minimal" RNase P RNAs.

A photoaffinity approach is being used to identify RNase P RNA residues that are located at or near the active site. UV irradiation of mature tRNA containing a photolabile azidophenacyl group on the 5' phosphate (the substrate phosphate) and bound to RNase P RNA under reaction conditions, results in the efficient crosslinking of the tRNA to RNase P RNA. Crosslinked nucleotides in the RNase P RNA, potentially involved in the reaction, were identified by primer extension. The analysis was carried out with RNase P RNA from 3 disparate eubacteria: B. subtilis, E. coli, and Chromatium vinosum. The same 2 discrete regions, of only a few nucleotides each, were crosslinked in each RNA. The crosslinked sequences are highly conserved and located within the core of the phylogenetic structure model.

A phylogenetic tree based on sequence alignments and signature elements of RNase P RNAs is nearly congruent with that derived from 16S rRNA sequence comparisons. The only difference in branching order seems to result from an unusually high rate of evolution of RNase P RNA sequence and structure in Alcaligenes eutrophus. This is in agreement with the previously observed relatively rapid rate of evolution of 16S rRNA in b-purple eubacteria.