JWB
James W. Brown

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

RNA Processing Meeting (the RNA Society), May 17-21, 1995, Cold Spring Harbor, NY

Focus on the Heart of Ribonuclease P

Norman R. Pace, James W. Brown, Jiunn-Laing Chen, Daniel N. Frank, Elizabeth S. Haas, Michael E. Harris, James M. Nolan, Bon-Kyeong Oh, Mary Anne Rubio, and Robert Siegel

Indiana University, Bloomington, IN 47405

Ribonuclease P removes 5' precursor sequences from pretRNAs. RNase P contains RNA and protein subunits; the RNA (400nt) is the catalytic moiety. Understanding the function of this ribozyme requires knowledge of its structure.

Comparative analysis of ca. 50 RNase P RNA sequences from diverse bacteria has resulted in a highly ordered secondary structure model; in the E. coli RNA, for instance, 64% of the nt are engaged in proven (two or more instances of covariation) base pairs (1). The model has recently been slightly refined and base-specific tertiary structure interactions identified by analysis of greater than 50 additional diverse sequences derived from naturally occurring microbial populations. A "phylogenetic minimum consensus structure", which includes only sequences and structures present in all instances of the RNA, illuminates the functional core of the ribozyme and identifies particularly conserved elements for experimental attention.

Photoaffinity crosslinking experiments are being used to gain further structural perspective on RNase P RNA. Arylazide photoagents are attached to various specific positions in RNase P RNA or tRNA, and sites of insertion upon irradiation are identified by primer extension. Modification of specific nt in the RNAs is accomplished by derivatization of 5' or 3' ends of circularly permuted RNAs (2). Analysis of greater than 100 crosslinking constraints in the context of the known structures of the RNAs, using a molecular mechanics-based refinement protocol, has resulted in an internally consistent model of the tertiary structure of the ribozyme-substrate complex (3), currently being refined on the basis of many new crosslinks.

Based on the structure model, conjugates (co-transcripts) of circularly permuted RNase P RNAs and tRNA have been fabricated so that the substrate is positioned at the active site of the ribozyme. These ribozyme-substrate conjugates undergo accurate intramolecular cleavage with a first-order rate equivalent to that of the chemical step of the native reaction (4). Such conjugates allow the separation of active and inactive ribozyme-substrate complexes, by size-change upon reaction, so can be used for modification-interference and in vitro selection experiments. The results of phosphorothioate modification-interference experiments identify non-bridging phosphate oxygens required for cleavage, likely Mg2+ -binding sites, in the catalytic heart of RNase P RNA.

References:

1. Haas, E.S. et al. (1994) Proc. Natl. Acad. Sci. USA 91:2527-2531.
2. Nolan, J.M. et al. (1993) Science 261:762-765.
3. Harris, M.E. et al. (1994) EMBO J. 13: 3953-3963.
4. Frank, D.N. et al. (1994) Biochemistry 33:10800-10808.

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