TOWARD THE STRUCTURE OF RIBONUCLEASE P.

Norman R. Pace, Amy B. Banta, James W. Brown, Elizabeth S. Haas, Michael E. Harris, Thomas E. LaGrandeur, James M. Nolan, Bong-Kyeong Oh and Mary Anne Rubio.
Department of Biology, Indiana University, Bloomington, IN 47405.

RNase P cleaves leader sequences from pre-tRNAs. In vivo RNase P is composed of protein and RNA. In vitro, at high ionic strength, the bacterial RNase P RNA is active in the absence of the protein moiety. Knowledge of the structure of RNase P RNA is crucial to understanding its action.

Ongoing phylogenetic-comparative analyses of bacterial RNase P RNA have recently refined the secondary structure model. In the current model of Escherichia coli RNase P RNA, for instance, 64% of its 377 nucleotides are engaged in proven helices. This is comparable to the extent of pairing in 16S rRNA (60%) or tRNAPhe (55%). Current comparative sequencing efforts seek to define the core of the ribozyme essential for catalysis, and to identify tertiary structure contacts. Analyses of RNase P RNAs from diverse (by rRNA criteria) organisms have revealed that some helical elements formerly thought to be universal in the phylogenetic domain of Bacteria in fact are dispensable. The theme emerging is that non-helical regions of the RNA, arranged and stabilized by helical elements, constitute the active site. Tertiary structure contacts are being sought by covariation analysis using a large data set of sequences, with less attention to phylogenetic diversity. Large numbers of partial RNase P RNA genes for sequencing are accumulated by PCR, using template DNA from complex, naturally occurring microbial populations.

A combination of molecular dynamics computer modeling, and inter- and intramolecular arylazide photoaffinity crosslinking is being used to orient RNase P RNA structural elements relative to one another, and thereby to infer a first-order tertiary structure model. Photoagents are attached to RNase P RNA or tRNA through a 5'-terminal thiophosphate, incorporated into T7 RNA polymerase transcripts by priming with guanosine monophosphorothioate, or to a 3'-terminus following chemical modification. Placement of the photoagent on specific nucleotides which are normally internal in the sequence is accomplished using circularly perinuted tRNA or RNase P RNA genes as templates for transcription. Sites of crosslinking of tRNA to RNase P RNA, determined by primer extension, outline the substrate binding site and, since the tertiary structure of tRNA is known, identify the three-dimensional positions of the crosslinked RNase P nucleotides. Sites of intramolecular crosslinking in RNase P RNA identify neighboring residues in the RNA and thereby establish constraints for computer modeling. Experiments are conducted with RNase P RNAs from multiple organisms in order to distinguish general from idiosyncratic results. The current version of the RNase P RNA tertiary structure will be presented.