Analysis of hairpin ribozyme base mutations in loops 2 and 4 and their effects on cis-cleavage in vitro.
In order to determine base requirements in loops 2 and 4 of the hairpin ribozyme, a comprehensive mutational analysis of the wild type sequence was done. Each base position in these two loops was mutated to contain each of the three non-wild type bases, and the effects of these mutations were analyzed using cis-cleavage assays. The method of data analysis allowed for the determination of self-cleavage rates as well as the fraction of transcripts produced which were uncleavable. Three positions in loop 2 (A22, A23, and C25) and one position in loop 4 (A38) resulted in no detectable self-cleavage when mutated to any of the non-wild type bases. The remainder of the base positions showed varying degrees of tolerance to base mutations with respect to their support of cis-cleavage. Evidence was obtained for the presence of a non-Watson-Crick base pair between A26 and G36 in the catalytic conformation of the hairpin ribozyme. On the basis of these results, a two-dimensional model for the hairpin ribozyme is presented.
Siwkowski
A
A
Department of Biological Sciences, Northern Illinois University, DeKalb 60115, USA.
Shippy
R
R
Hampel
A
A
D001482:Base Composition; D001483:Base Sequence; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D016337:RNA, Catalytic; D012313:RNA