MUTATIONAL ANALYSIS OF SACCHAROMYCES-CEREVISIAE NUCLEAR RNASE-P - RANDOMIZATION OF UNIVERSALLY CONSERVED POSITIONS IN THE RNA SUBUNIT

Three regions in the Saccharomyces cerevisiae RNase P RNA have been id entified, at positions See 87-94, See 309-316, and See 339-349, that c ontain nucleotides that are invariant in identity and position among a ll the known RNase P RNAs. To study the importance of these conserved RPR1 RNA regions in enzyme function, three independent mutational libr aries were created in which the positions of invariant nucleotides wer e randomized simultaneously. Screening in vivo was used to identify vi able RPR1 variants when reconstituted into holoenzyme in cells. Despit e the universal evolutionary conservation, most of these positions tol erate certain sequence changes without severely affecting function. Mo st changes, however, produced subtle defects in cell growth and RNase P function, supporting the importance of these conserved regions. Isol ation of conditional growth mutants allowed the characterization of th e effects of mutations on cell growth, RPR1 RNA maturation, and activi ty of the holoenzyme in vitro. Kinetic analysis showed that viable var iants were usually more defective in catalytic rate (k(cat)) than in s ubstrate recognition (K-m).