1992 RNA Processing Meetings, Keystone, CO

TOWARD STRUCTURE AND MECHANISM OF RIBONUCLEASE P.

Norman R. Pace, James W. Brown, Elizabeth S. Haas, James M. Nolan, Bong-Kyeong Oh, and Drew Smith. Department of Biology, Indiana University, Bloomington, IN 47405.

RNase P cleaves leader sequences from pretRNAs. 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. The secondary structure of the bacterial RNA is increasingly well-defined by phylogenetic comparative analysis.

A photoaffinity crosslinking approach is being used to identify structural elements in RNase P RNA that are proximal to an arylazide photoagent at different positions in the tRNA substrate. The arylazide is attached to 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 permuted tRNA genes as templates for transcription. Depending on the position of the photoagent in the tRNA, UV-induced crosslinking to RNase P RNA occurs with variable efficiency. Sites of crosslinking, identified by primer extension, are expected to be in the vicinity of the substrate-binding site. Crosslinks between the normal 5'-terminus of mature tRNA (the substrate phosphate) and RNase P RNA identify residues in the vicinity of the active site of the ribozyme. Since the three-dimensional structure of tRNA is known, the results point to the three-dimensional positions of the crosslinked nucleotides in RNase P RNA.

The dependence of the RNase P reaction on divalent cation, pH and chemical alteration of the precursor nucleotide adjacent to the cleaved phosphate have been investigated by detailed kinetic analyses. Divalent cation is required for the mechanism of catalysis, not for some structural aspect of the enzyme or substrate RNAs. Maximum rate of ribozyme action requires three Mg2+ ions. One interpretation of the pH-dependence of the RNase P reaction is that hydroxide is a co-substrate in the reaction: dependence upon [OH-] is Michaelean. Manipulation of reaction pH therefore allows manipulation of kchem, the rate of the hydrolytic step. At pH 8, kchem of the RNase P reaction is approximately 180 min- 1. This rate is much higher than kcat of the overall reaction (0.5 min-1 for RNA- alone, 20 min-1 for holoenzyme), which is rate-limited by dissociation of the enzyme product complex. This difference in kchem and kcat is a significant consideration in the search for mutants in RNase P RNA that have impact on mechanism: major damage to kchem may not be reflected in kcat as measured in the usual assays.

The catalytic step of the RNase P reaction depends upon the 2'-OH of the precursor nucleotide adjacent to the cleaved phosphodiester. Conversion of that -OH to 2'-deoxy reduces kchem 104-fold. Considerable activity is recovered by increase in [Mg2+], indicating that the vicinal 2'-OH participates in binding Mg2+ (hydrate). Conversion to 2'-O-methyl reduces kchem 106-fold beyond the rate of cleavage of the native substrate. These results are interpreted in terms of a discrete mechanism of action of RNase P.