James W. Brown

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

Third International Symposium on Catalytic RNAs (Ribozymes) and Targeted Gene Therapy of the Treatment of HIV Infection, San Diego, CA.1992

Characterization of RNase P RNAs from Thermophilic Bacteria.

James W. Brown, Elizabeth S. Haas, and Norman R. Pace, Department of Biology and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, IN 47405

The 5´-leader sequences of precursor tRNA molecules are removed endonucleolytically by ribonuclease P (RNase P). In Bacteria, RNase P is composed of a large (ca. 400nt) RNA and a relatively small (ca. 14Kd) protein. Although the enzyme functions in vivo as a ribonucleoprotein, the RNA alone is capable in vivo of accurately cleaving precursor tRNAs in the presence of elevated salt concentrations.

Because the RNAs from thermophilic Bacteria necessarily function at very high temperatures, their global packing energies are likely to be greater than the corresponding RNAs from mesophilic organisms. Consequently, RNAs from thermophiles are likely to be more resistant to destabilizing factors that might be introduced by synthetic changes. These RNAs, therefore, are potentially useful starting points for the engineering of functional, minimal RNase P RNAs. The properties of the RNase P RNAs of the thermophilic Bacteria Thermotoga maritima and Thermus aquaticus are therefore being investigated with the aim of understanding and comparing the structural features of these RNAs that allow them to function at the high growth-temperatures of these organisms.

The RNase P RNAs of the thermophilic organisms (transcribed in vitro) are inherently thermostable. The Tms of the RNase P RNAs from both thermophiles at low ionic strength (10mM Na+) are 9C higher than the E. coli RNA. At high ionic strength (1000mM Na+), the difference in Tms of the thermophilic RNAs relative to E. coli RNA is ca. 18C. Cleavage rates of precursor tRNAs, in the RNA-alone reaction, by the RNase P RNAs of Thermus and Thermotoga are optimal at 50 - 60C in the presence of 1M NH4Cl, 5 - 10C higher than that of E. coli (a mesophile) RNase P RNA under the same conditions. At optimal salt concentrations (3 - 5M NH4Cl), the temperature optima of the thermophilic RNAs increase by ~5C. The Mg++ requirement of the thermophilic RNAs is similar to that of the E. coli RNA. Polyamines (spermine or spermidine) have little or no effect on reaction rate. Activity in low salt (100mM NH4Cl) can be recovered with either of the thermophilic RNase P RNAs in the presence of the protein component of E. coli RNase P.

Several features of these thermophilic RNase P RNAs suggest mechanisms by which at least some degree of thermostability might be attained. Although the overall G+C contents of the thermophilic RNAs are only slighly higher than their mesophilic counterparts, there is a 12 - 16% increase in G-C pairs, and non-Watson-Crick base-pairs (G=U, A=C, G=A) are rare. In addition, the RNA from Thermotoga is shorter than any other known bacterial RNase P RNA (338nt), which may stabilize the structure by minimizing the number of folding alternatives. Some nucleotides are not present in highly constrained regions of the RNA , which may stabilize the RNA to the effects of thermal vibration.

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