Nucleic Acids Research, 1994, Vol. 22, No. 6 1096-1100
© 1994
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Mutagenesis of the hairpin ribozyme
Departments of Biological Sciences and Chemistry, Northern Illinois University DeKalb, IL 60115 1SRI International 333 Ravenswood Avenue, Menlo Park, CA 94025 2Department of Chemistry, University of California Berkeley, CA 94720, USA
*To whom correspondence should be addressed
Received September 28, 1993. Revised January 22, 1994. Accepted January 22, 1994.
Extensive in vitro mutagenesis studies have been performed on the hairpin ribozyme and substrate in an effort to refine the overall secondary structure of the molecule and provide further insight into what elements are essential for activity. A secondary structure consisting of four helices and five loop regions remains the basic model as originally proposed. Two helices, helix 1 and 2, form between the substrate and ribozyme while helices 3 and 4 are within the ribozyme itself. Our results suggest that hellces 3 and 4 are smaller than previously proposed, consisting of four base pairs and three base pairs respectively. Helix 4 can be extended without loss of activity and loop 3 at the closed end of the hairpin model can be varied in sequence with retention of activity. There is an unpaired nucleotide between helices 2 and 3 consisting of a single A base, suggesting the opportunity for flexibility within the tertiary structure at this point. Comparisons are made between the new data and previously published mutagenesis and phylogenetic data. Substrate targeting rules require base pairing between helices 1 and 2 with cleavage (*) occurring in a preferred 5'(g/c/u)n*guc3' sequence of the substrate.
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