JWB
James W. Brown

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

1991 Cold Spring Harbor RNA Processing Meeting, Cold Spring Harbor, N.Y.

TOWARD THE STRUCTURE AND MECHANISM OF RIBONUCLEASE P

Norman R. Pace, James W. Brown, Alex B. Burgin, Sylvia C. Darr, Elizabeth S. Haas, Dirk A Hunt, Drew Smith and Karen Zito Department of Biology, and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, IN 47405

RNase P cleaves leader sequences from pre-tRNAs. 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 monovalent and divalent cation concentrations, the RNA alone is an efficient and accurate catalyst.

The secondary structures of the eubacterial RNase P RNAs are being elucidated using a phylogenctic comparative approach to identify base pairs. Variation among known RNase P RNAs is substantially due to the presence or absence of discrete structural domains distributed in a conserved core of homologous sequence and secondary structure. It is clear that the conserved core contains the RNase P activity. A synthetic RNasc P RNA (Min I RNA), consisting of only the conserved structure (263 nt), is highly active. However, Min1 RNA has some properties that differ from the native RNA: increased (100X) Km, requirement for higher ionic strength for activity, and reduced thermal stability. The structural basis of these differences has been traced to three of the four sequence alterations made in the E. coli RNA in constructing Min1 RNA. The phenotypic effects of each of the deleterious alterations are additive in Min 1 RNA.

A photoaffinity approach was 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) is highly efficiently crosslinked to RNase P RNA under reaction conditions. Cross-linked nucleofides in the RNase P RNA, potentially involved in the reaction, were identified in RNase P RNAs from three disparate eubacteria: B. subtilis, Chromatium vinosum and E. coli. The same two discrete regions, in the core of the phylogenetic structure model, were crosslinked in each type of RNA. The crosslinking reaction also is a sensitive assay for substrate-binding that is independent of catalytic activity.

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. At high monovalent ionic strength, in the absence of divalent cations, RNase P RNA - tRNA crosslinks can form as efficiently as in the presence of Mg2+, and 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, and a concentration-dependence for cleavage of a deoxynucleotide-containing substrate by RNase P, provide evidence for a discrete mechanism of action of the ribozyme.

nullLast updated by James W Brown