JWB
James W. Brown

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

1993 7th ISSOL Meeting, Barcelona, Spain

Structure and evolution of an RNA enzyme: Ribonuclease P.

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, USA

Ribonuclease P (RNase P) cleaves leader sequences from precursors of transfer RNA (tRNA) to generate the mature 5´ end of the tRNA (1). Bacterial RNase P occurs in vivo as a complex containing a small protein (ca. 14Kd in Escherichia coli) and a much larger RNA (ca. 130Kd). At high ionic strength, in vitro, the RNA alone is an efficient and accurate catalyst; it is the only known naturally-occuring RNA that functions as an enzyme, in the sense that each molecule of RNase P RNA processes many substrate molecules. Knowledge of the mechanisms of tRNA recognition and catalysis by RNase P RNA is therefore an essential prerequisite to further understanding of the 'RNA world'.

The structure of RNase P RNA is being examined by phylogenetic comparative analysis. Complete and partial RNase P RNA gene sequences from representatives of nine evolutionarily distant phylogenetic groups of Bacteria (37 sequences in all) have so far been determined; these sequences have been used to develop a high-resolution model for the secondary structure of the RNA (2). Although the gene sequences are only 35 - 55% identical, the secondary structures of the encoded RNAs are highly conserved (Figure). However, the structure of RNase P RNA in the low G+C Gram-positve bacteria (such as Bacillus) is significantly altered; several large insertions and deletions have occurred in otherwise conservative regions of the molecule. The minimum conserved structure present in both the low G+C Gram positive bacteria and other organisms has nevertheless been shown to contain all of the elements essential for catalysis (3); the roles of the dispensable elements are currently under investigation. Analysis of one of the variably-present elements suggests that, in much the same way that the secondary structures are more conservative than the primary sequences, the tertiary structures of the Bacillus and E. coli RNAs may more closely resemble one another than the secondary structures would imply (4).

The RNase P enzymes of the Archaea (formerly archaebacteria) contain RNA components with striking similarity to those of the Bacteria. In contrast to their bacterial homlogs, the archaeal RNase P RNAs are dependent on the protein components of the enzyme for activity in vitro. Although the RNase P RNA gene sequences are so far only available from extreme halophiles (5,6) and Sulfolobus solfataricus (7), the emerging secondary structures of these RNAs are remarkably similar to those of the Bacteria. The archaeal RNAs contain all of the structural elements found in the bacterial minimum consensus model, including even the identities of the majority of invariant bacterial nucleotides. The reason for the dependence of the archaeal RNase P activity on protein components is therefore unclear. It appears that in contrast to the bacterial enzyme, at least in the case of S. solfataricus, the enzyme is predominantly protein rather than RNA. These protein components have yet to be examined. RNase P may therefore serve as a model for the aquisition of RNA function by protein, a process demanded by the RNA world hypothesis about which little is known.

The tertiary structure of RNase P RNA is being examined by a combination of comparative and experimental methods. Co-axial stacking of helices in the secondary structure have been identified by comparative analysis; the relative positions and orientations of these stacks are being determined in photoaffinity crosslinking experiments. Models for the three-dimensional structure of the RNA are being constructed by a combination of molecular mechanics and manual methods, which are evaluated on the basis of known phylogenetic variation in RNase P RNA structure.

1. Darr, S.D.,J.W.Brown, and N.R.Pace. TIBS (1992) 17:178-182.
2. Brown, J.W. and N.R.Pace. Nucl. Acids Res. (1992) 20:1451-1456.
3. Waugh, D.S., C.J.Green, and N.R.Pace. Science (1989) 244:1569-1571.
4. Haas, E.S., D.P.Morse, J.W.Brown, F.J.Schmidt, and N.R.Pace. Science (1991) 254:853-856.
5. Neuwlandt , D.T., E.S.Haas, and C.J.Daniels. J. Biol. Chem. (1991) 266:5689-5695.
6. Armburster, D., E.S.Haas, J.W.Brown and C.J.Daniels (unpublished)
7. LaGrandeur, T., S.D.Darr, E.S.Haas, and N.R.Pace. (submitted)

Figure. Line representations of RNase P RNA secondary structure.

nullLast updated by James W Brown