ANALYSIS OF THE CYT-18 PROTEIN-BINDING SITE AT THE JUNCTION OF STACKED HELICES IN A GROUP-I INTRON RNA BY QUANTITATIVE BINDING ASSAYS AND IN-VITRO SELECTION

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 pr otein) functions in splicing group I introns by promoting the formatio n of the catalytically active structure of the intron RNA. Previous st udies showed that CYT-18 binds with high affinity to the P4-P6 domain of the catalytic core and that there is some additional contribution t o binding from the P3-P9 domain. Here, quantitative binding assays wit h deletion derivatives of the N. crassa mitochondrial large rRNA intro n showed that at least 70% of the binding energy can be accounted for by the interaction of CYT-18 with the P4-P6 domain. Within this domain , P4 and P6 are required for high affinity CYT-18 binding, while the d istal elements P5 and P6a may contribute indirectly by stabilizing the correct structure of the binding site in P4 and P6. CYT-18 binds to a small RNA corresponding to the isolated P4-P6 domain, but not to a pe rmuted version of this RNA in which P4-P6 is a continuous rather than a stacked helix. Iterative in vitro selection experiments with the iso lated P4-P6 domain showed a requirement for base-pairing to maintain h elices P4, P6 and P6a, but indicate that P5 is subject to fewer constr aints. The most strongly conserved nucleotides in the selections were clustered around the junction of the P4-P6 stacked helix, with ten nuc leotides (J3/4-2,3, P4 bp-1 and 3, and P6 bp-1 and 2) found invariant in the context of the wild-type RNA structure. In vitro mutagenesis co nfirmed that replacement of the wild-type nucleotides at J3/4-2 and 3 or P4 bp-3 markedly decreased CYT-18 binding, reflecting either base s pecific contacts or indirect readout of RNA structure by the protein. Our results suggest that a major function of CYT-18 is to promote asse mbly of the P4-P6 domain by stabilizing the correct geometry at the ju nction of the P4-P6 stacked helix. The relatively large number of cons erved nucleotides at the binding site suggests that the interaction of CYT-18 with group I introns is unlikely to have arisen by chance and could reflect either an evolutionary relationship between group I intr ons and tRNAs or interaction with a common stacked-helical structural motif that evolved separately in these RNAs. (C) 1996 Academic Press L imited