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 sequence conservation only in domain IV, a bulged hairpin capped by a GNRA (N, any nucleotide; R, purine) tetranucleotide loop except in plant homologs. Tetraloops conforming to this sequence or to the consensus UNCG enhance the stability of synthetic RNA hairpins and have strikingly similar three-dimensional structures. To determine the biological relevance of this similarity, as well as to assess the relative contributions of sequence and structure to the function of the domain IV tetraloop, we replaced the GAAA sequence in fission yeast SRP RNA with UUCG. Haploid strains harboring this substitution are viable, providing experimental evidence for the functional equivalence of the two tetraloops. We next tested the two sequences found in plant SRP RNAs at this location for function in the context of the Schizosaccharomyces pombe RNA. While substitution of CUUC does not allow growth, a viable strain results from replacing GAAA with UUUC. Although the viable tetraloop substitution 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 substitutions exhibit subtle differences from the wild type not only in the tetraloop itself, but also in the 3-base pair adjoining stem. To directly assess the importance of the latter structure, we disrupted it partially or completely and made the compensatory mutations to restore the helix. Surprisingly, mutant RNAs with as little as one Watson-Crick base pair can support growth.
Selinger
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Department of Biochemistry, University of Illinois, Urbana-Champaign 61801.
Liao
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Wise
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JA
D000483:Alleles; D001482:Base Composition; D001483:Base Sequence; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D010957:Plasmids; D017354:Point Mutation; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D018271:Signal Recognition Particle; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D012331:RNA, Fungal; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle