RNA 96 (the RNA Society), May 28 - June 2 1996, Madison, WI

RNase P RNA from Mycoplasma fermentans Simplifies our View of the Catalytic Core of the Ribozyme

Robert W. Siegel*, Amy B. Banta*, Elizabeth S. Haas**, James W. Brown**, and Norman R. Pace*
*
Department of Biology and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, IN 47405
**Department of Microbiology, North Carolina State University, Raleigh, NC 27695

The catalytic RNA moiety of (eu)bacterial RNase P is responsible for cleavage of the 5' leader sequence from precursor tRNAs. We report a sequence, phylogenetic-comparative structural analysis, and kinetic characterization of the RNase P RNA from Mycoplasma fermentans, at 276 nucleotides the smallest known RNase P RNA. This RNA lacks a stem-loop structure (helix P12) which previously was though to be universally present in bacterial RNase P RNAs. Since structural elements not present in all instances of the ribozyme are not expected to be crucial for function, the absence of helix P12 from the M. fermentans RNase P RNA suggests that this structural element is not required for catalytic activity in vivo. In order to test this possibility in vitro, the kinetic properties of M. fermentans RNase P RNA and a mutant Escherichia coli RNase P RNA which was engineered to lack helix P12 were determined. These RNase P RNAs are catalytically active with efficiencies (kcat/Km) comparable to that of native E. coli RNase P RNA. These results show that helix P12 is dispensable in vivo in some organisms, and therefore must not be essential for the mechanism of RNase P action.

On the other hand, the blocks of sequence and structure that are present in every instance of bacterial RNase P RNA, constituting a "phylogenetic-minimum" core structure, are potentially involved in substrate binding and/or catalysis. In order to test the notion that the minimum structure is catalytically active and, conversely, that all phylogenetically volatile structures in RNase P RNA are dispensable for function, a simplified RNA representing the phylogenetic-minimum RNase P RNA was constructed by deleting all evolutionarily variable structures from the M. fermentans RNA. We show that this synthetic RNA (Micro P RNA) is catalytically active in vitro with approximately 600-fold decrease in catalytic efficiency relative to the native RNA.