1992 RNA Processing Meetings, Keystone, CO

Characterization of RNase P RNAs from Thermophilic Bacteria.

James W. Brown, Elizabeth S. Haas, and Norman R. Pace
Department of 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 ~400nt RNA and a relatively small (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. As part of an on-going phylogenetic characterization of RNase P RNA structure, we have investigated the genes encoding the RNase P RNAs of the thermophilic Bacteria Thermus aquaticus and Thermotoga maritima.

It is thought that the ancestor of the Bacteria, Archaea, and Eucarya was thermophilic, and that life may have originated at temperatures we now consider to be high. Thermophilicity in modern organisms is therefore thought to be "ancestral" (primitive) in some cases and "recent" (derived) in others. The characteristics which impart thermal stability to macromolecules from ancestral and derived thermophilic organisms may be quite different. The properties of the RNase P RNAs of Thermotoga maritima (an ancestral thermophile) and Thermus aquaticus (likely a derived thermophile) are 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. 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 in the case of the Thermus RNase P RNA, but not the Thermotoga RNA, in the presence of the protein component of E. coli RNase P. Thermal-denaturation experiments are being used to study the thermostability of these RNAs independently of enzymatic activity.

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 content 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, U=U) 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. Also, some nucleotides are not present in highly constrained regions of the RNA , which may stabilize the RNA to the effects of thermal vibration. It seems likely, however, that other factors, such as the protein component(s), base modifications, cellular matrix associations, and chemical environment, also strongly influence the thermostability of these RNAs in vivo.