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.

Phylogenetic analysis of the higher-order structure of eubacterial RNase P RNA.

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 eubacteria, the catalytic component of RNase P is a ~400nt RNA; the relatively small (14Kd) protein component is dispensable in vitro.

The structure of eubacterial RNase P RNA is being elucidated using the phylogenetic comparative approach. The genes encoding the RNase P RNA from Deinococcus radiodurans (444nt), Thermus aquaticus (369nt), Thermotoga maritima (338nt), and Thermotoga neapolitana (338nt) have recently been cloned and sequenced. These organisms represent two evolutionary lines of descent ("phyla") which are very divergent from the main group of eubacteria, including the two lineages from which RNase P RNA sequences were previously available (the proteobacteria and Gram positive eubacteria). In addition, these organisms, with the exception of D. radiodurans, are the most extremely thermophilic eubacteria known, with optimal growth temperatures of ~75C and maximum growth tempertures of up to 90C. Analysis of the available eubacterial RNase P RNA sequences are being used to further expand and refine the model for secondary and higher-order structure of eubacterial RNase P RNA.

One such addition is the identification of a four base-pair helix formed by the pairing of nucleotides 82-85 and 276-279 (numbering based on the Escherichia coli sequence), which generates a second pseudoknot in the RNase P RNA structure model. The presence of this helix is supported by compensatory base substitutions in three of the four potential pairings. The pairing of nucleotides 83 and 278 is particulary well supported - these nucleotides have changed in concert at least three times in the four eubacterial phyla for which sequences are available. The pairing of nucleotides 82-85/276-279 suggests that the absence of helix 270-274/280-284 in the Bacillus RNase P RNAs may be compensated for structurally by the presence of a group-specific helix at position 80-81; the pseudoknot brings helix 270-274/280-284 into a position similar to that of the group-specific helix 80-81 in Bacillus. Working tertiary models of eubacterial RNase P RNA are being constructed based on the refined secondary structure model, using the topological constraints on RNase P RNA structure imparted by the presence of the two pseudoknots, steric constraints, cross-linking information, available chemical and enzymatic structure probing information, and other data.

The new RNase P RNAs are strikingly similar in secondary structure to those of the proteobacteria. The only novel group-specific element found is a 43nt extension to helix 108-110/115-117 in D. radiodurans; none of the group-specific elements of the Bacillus (Gram positive) structures are present. Because Thermotoga, Thermus, and Deinococcus, along with the green non-sulfur eubacteria, represent the deepest known divergences in the eubacteria, the similarity in the structures of the RNase P RNAs from these organisms and the proteobacteria indicate that this structural "plan" is ancestral to the entire eubacterial lineage. The drastic differences in the structure of RNase P RNA in Bacillus probably indicate that an episode of very rapid change in RNase P RNA occurred at some point in the evolution of the low G+C branch of the Gram positive eubacteria. A less drastic example of "punctuated equilibrium" seems also to have occurred in the line of descent leading to the b- proteobacterium Alcaligenes eutrophus, in which, like Bacillus, two highly conservative helices have been lost.

nullLast updated by James W Brown