Sequence Requirements of the Hammerhead RNA Self-Cleavage Reaction

Duane E. Ruffner, Olke C. Uhlenbeck, Gary D. Stormo

Research output: Contribution to journalArticle

430 Scopus citations

Abstract

A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study. The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined. Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage. In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex. In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing an alternate substrate or ribozyme secondary structure. All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA. The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences.

Original languageEnglish
Pages (from-to)10695-10702
Number of pages8
JournalBiochemistry
Volume29
Issue number47
DOIs
StatePublished - Nov 1 1990
Externally publishedYes

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