James W. Brown

Associate Professor & Undergraduate Coordinator
Department of Microbiology, NC State University

1991 Keystone Symposia on Molecular and Cellular Biology, Keystone, CO


Norman R. Pace, James W. Brown, Alex B. Burgin, Sylvia C. Darr, Elizabeth S. Haas, Dirk A Hunt and Drew Smith

Department of Biology, Indiana University, Bloomington, IN 47405.

RNase P cleaves leader sequences from pretRNAs. In the eubacteria Bacillus subtilis and Escherichia coli, RNase P is composed of protein (119 amino acids) and RNA (ca. 400 nucleotides). In vitro, at high salt concentrations, the RNA alone is an efficient and accurate catalyst. The high salt (or RNase P protein) is thought to screen electrostatic repulsion between enzyme and substrate RNAs.

The secondary structures of the eubacterial RNase P RNAs are being elucidated using a phylogenetic comparative approach to test base pairing possibilities. Variation among known RNase P RNAs is substantially due to the presence or absence of discrete structural domains scattered in a highly conserved core of homologous sequence and secondary structure. It is clear that the conserved core contains the RNase P activity: a synthetic RNase P RNA, consisting of only the conserved structure (263 nt), has nearly native activity. Comparative analysis of RNase P RNAs from more diverse organisms provides further perspective on the conserved core of the RNA and identifies structural features useful for designing new synthetic RNase P RNAs.

A photoaffinity approach is being used to identify RNase P RNA residues that are located at or near the catalytically active site. A mature tRNA containing a photolabile azidophenacyl group on the 5'phosphate (the substrate phosphate) was bound to RNase P RNA under reaction conditions. UVirradiation resulted in highefficiency 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 has been carried out with RNase P RNAs from three disparate eubacteria: B. subtilis, Chromatium vinosum and E. coli. The same two discrete regions, of only a few nucleotides each, were crosslinked in each type of RNA. The crosslinked sequences are highly conserved and located in the core of the phylogenetic structure model.

The action of RNase P requires divalent cations: Mg2+, Mn2+ or Ca2+. There has been no evidence to distinguish whether divalent. cations are required for the structure of the RNA, or for the catalytic mechanism. The possible structural role for divalent cations was tested by crosslinking experiments as described above. At high monovalent ionic strength, in the absence of divalent cations, RNase P RNA tRNA crosslinks can form, in some instances nearly as efficiently as in the presence of Mg2+. The sites of crosslinking are the same in the presence or absence of Mg2+. These observations suggest that the global and local conformations of the enzyme and substrate RNAs are proper for catalytic function in the absence of divalent cations. The absolute requirement for divalent cations for catalysis by RNase P therefore indicates that divalent cations are intrinsic to the catalytic mechanism.

Substitution of deoxyribose instead of ribose, at the pretRNA site cleaved by RNase P reduces the rate of cleavage by 103 to 104 -fold. A model for the mechanism of RNase P action and the involvement of the substrate 2'OH has been developed.

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