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 eubac terium Thermus thermophilus HB8. Catalytic activity was reduced 600-fo ld when the terminal helix, connecting the 5' and 3' ends of the molec ule, was destroyed by deleting 15 nucleotides from the 3' end. In comp arison, the removal of a large portion (94 nucleotides, about one quar ter of the RNA) of the upper loop region impaired function only to a r elatively moderate extent (400-fold reduction in activity). The termin al 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 stru cture was shown to be a key element for enzyme function, as was the re gion of nucleotides 328-335. Deleting a conserved hairpin (nucleotides 304-327) adjacent to this region and replacing the hairpin by a tetra nucleotide sequence or a single cytidine reduced catalytic activity on ly 6-fold, whereas a simultaneous mutation of the five highly conserve d nucleotides in the region of nucleotides 328-335 reduced catalytic a ctivity by >10(5)-fold. The two strictly conserved adenines 244 and 24 5 (nucleotides 248/249 in Escherichia coil RNase P RNA) were not as es sential for enzyme function as suggested by previous data. However, ad ditional disruption of two helical segments (nucleotides 235-242) adja cent to nucleotides 244 and 245 reduced activity by >10(4)-fold, suppo rting the notion that nucleotides in this region are also part of the active core structure. Finally, we suggest that the severe interferenc e with RNase P RNA function caused by many deletions supports the noti on that the great bulk of its almost 400 nucleotides co-organize a sin gle domain of higher order RNA structure.