RNASE P RNA TERTIARY INTERACTIONS IDENTIFIED BY ANALYSIS OF SEQUENCES FROM MIXED NATURAL MICROBIAL POPULATIONS

J. W. Brown1, E. S. Haas1, M. A. Rubio2, and N. R. Pace2

1 Department of Microbiology, North Carolina State University, Raleigh, NC 27695 USA
2 Department of Biology, Indiana University, Bloomington, IN 47405 USA

Comparative sequence analysis has proven to be the most generally useful approach to examining the structures of natural RNAs. Typically, sequences are obtained one-at-a-time from isolated species, but for comparative analyses the number of different sequences available for analysis is more important than knowledge of the source of those sequences. As part of an on-going comparative study of RNase P RNA structure, we have developed a PCR-based method for rapidly obtaining large sequence collections using DNA from mixed naturally-occurring microbial populations expected to contain thousands of different bacterial species.

DNA isolated from lake sediment, pond water filtrate, and hot-spring biomass was used as template in PCR reactions using primers complementary to highly-conserved sequences near the 5' and 3' ends of bacterial RNase P RNA-encoding genes. 49 unique clones were sequenced, more than doubling the collection of bacterial RNase P RNA sequences available. This enlarged sequence collection has been used to refine the current secondary structure model, and in a search for tertiary interactions.

The sequences provide evidence requiring minor adjustment of the central region of helix P12 (nucleotides 142-176) of the E. coli RNA, and against pairing of nucleotides 304:327 and 305:326 in helix P18. There are no remaining unpaired, phylogenetically consistent dinucleotide complements in the sequence alignments; all of the conserved secondary structure in RNase P RNA has been detected.

Two tertiary interactions have also been identified in this analysis. In both cases, the interaction indicated is a base-triple formed by the 'R' of a GNRA tetraloop interacting with the purine of a standard base-pair, such that the base-triple is either A:G=C or G:A=U. Modeling studies suggest that the third base interacts with the basepair in the minor groove of the helix, as has been suggested for similar covariations in group I intron RNAs.