IN-VITRO SELECTION OF RNASE-P RNA REVEALS OPTIMIZED CATALYTIC ACTIVITY IN A HIGHLY CONSERVED STRUCTURAL DOMAIN

In vitro selection techniques are useful means of dissecting the funct ions of both natural and artificial ribozymes. Using a self-cleaving c onjugate containing the Escherichia coli ribonuclease P RNA and its su bstrate, pre-tRNA (Frank DN, Harris ME, Pace NR, 1994, Biochemistry 33 :10800-10808), we have devised a method to select for catalytically ac tive variants of the RNase P ribozyme. A selection experiment was perf ormed to probe the structural and sequence constraints that operate on a highly conserved region of RNase P: the J3/4-P4-J2/4 region, which lies within the core of RNase P and is thought to bind catalytically e ssential magnesium ions (Harris ME et al., 1994, EMBO J 13:3953-3963; Hardt WD et al., 1995, EMBO J 14:2935-2944; Harris ME, Pace NR, 1995, RNA 1:210-218). We sought to determine which, if any, of the nearly in variant nucleotides within J3/4-P4-J2/4 are required for ribozyme-medi ated catalysis. Twenty-two residues in the J3/4-P4-J2/4 component of R Nase P RNA were randomized and, surprisingly, after only 10 generation s, each of the randomized positions returned to the wild-type sequence . This indicates that every position in J3/4-P4-J2/4 contributes to op timal catalytic activity. These results contrast sharply with selectio ns involving other large ribozymes, which evolve improved catalytic fu nction readily in vitro (Chapman KB, Szostak JW, 1994, Curr Opin Struc t Biol 4:618-622; Joyce GF, 1994, Curr Opin Struct Biol 4:331-336; Kum ar PKR, Ellington AE, 1995, FASEB J 9:1183-1195). The phylogenetic con servation of J3/4-P4-J2/4, coupled with the results reported here, sug gests that the contribution of this structure to RNA-mediated catalysi s was optimized very early in evolution, before the last common ancest or of all life.