FUNCTIONAL INTERCHANGEABILITY OF THE STRUCTURALLY SIMILAR TETRANUCLEOTIDE LOOPS GAAA AND UUCG IN FISSION YEAST SIGNAL RECOGNITION PARTICLE RNA

Signal recognition particle (SRP) RNA exhibits significant primary seq uence conservation only in domain IV, a bulged hairpin capped by a GNR A (N, any nucleotide; R, purine) tetranucleotide loop except in plant homologs. Tetraloops conforming to this sequence or to the consensus U NCG enhance the stability of synthetic RNA hairpins and have strikingl y similar three-dimensional structures. To determine the biological re levance of this similarity, as well as to assess the relative contribu tions of sequence and structure to the function of the domain IV tetra loop, we replaced the GAAA sequence in fission yeast SRP RNA with UUCG . Haploid strains harboring this substitution are viable, providing ex perimental evidence for the functional equivalence of the two tetraloo ps. We next tested the two sequences found in plant SRP RNAs at this l ocation for function in the context of the Schizosaccharomyces pombe R NA. While substitution of CUUC does not allow growth, a viable strain results from replacing GAAA with UUUC. Although the viable tetraloop s ubstitution mutants exhibit wild-type growth under normal conditions, all three express conditional defects. To determine whether this might be a consequence of structural perturbations, we performed enzymatic probing. The results indicate that RNAs containing tetraloop substitut ions exhibit subtle differences from the wild type not only in the tet raloop itself, but also in the 3-base pair adjoining stem. To directly assess the importance of the latter structure, we disrupted it partia lly or completely and made the compensatory mutations to restore the h elix. Surprisingly, mutant RNAs with as little as one Watson-Crick bas e pair can support growth.