Contribution of structural elements to Thermus thermophilus ribonuclease P RNA function.

We have performed a deletion and mutational analysis of the catalytic ribonuclease (RNase) P RNA subunit from the extreme thermophilic eubacterium Thermus thermophilus HB8. Catalytic activity was reduced 600-fold when the terminal helix, connecting the 5' and 3' ends of the molecule, was destroyed by deleting 15 nucleotides from the 3' end. In comparison, the removal of a large portion (94 nucleotides, about one quarter of the RNA) of the upper loop region impaired function only to a relatively moderate extent (400-fold reduction in activity). The terminal helix appears to be crucial for the proper folding of RNase P RNA, possibly by orientating the adjacent universally conserved pseudoknot structure. The region containing the lower half of the pseudoknot structure was shown to be a key element for enzyme function, as was the region of nucleotides 328-335. Deleting a conserved hairpin (nucleotides 304-327) adjacent to this region and replacing the hairpin by a tetranucleotide sequence or a single cytidine reduced catalytic activity only 6-fold, whereas a simultaneous mutation of the five highly conserved nucleotides in the region of nucleotides 328-335 reduced catalytic activity by > 10(5)-fold. The two strictly conserved adenines 244 and 245 (nucleotides 248/249 in Escherichia coli RNase P RNA) were not as essential for enzyme function as suggested by previous data. However, additional disruption of two helical segments (nucleotides 235-242) adjacent to nucleotides 244 and 245 reduced activity by > 10(4)-fold, supporting the notion that nucleotides in this region are also part of the active core structure.(ABSTRACT TRUNCATED AT 250 WORDS)