Revised Manuscript Received August 7, 1990 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 conservedbase 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. e RNA genomes of several virusoids contain a domain of approximately 50 nucleotides that is required for an auto-catalytic cleavage step in their replication pathway (Forster & Symons, 1987a). A “hammerhead” secondary structure composed of three helices joined at a central core of 11-12 single-stranded nucleotides has been identified as necessary and sufficient for theself-cleavage reaction (Uhlenbeck, 1987; Forster & Symons, 1987b). The helices are stable enough to permit theassembly of active hammerheads from two or even three separate RNA molecules (Uhlenbeck, 1987; Haseloff & Gerlach, 1988; Koizumi et al., 1988; Jeffries & Symons, 1989), and catalytic cleavage can be demonstrated through successive cycles of helixannealing and dissociation (Uhlen-beck, 1987; Haseloff & Gerlach, 1988; Jeffries & Symons, 1989; Koizumi et al., 1989). This system is the smallest example of a catalytic RNA found to date. It is clear that the hammerhead musthave a fairly precise sequence requirement for cleavage. All three helices and thirteen conserved nucleotides appear in the seven available natural examples of hammerheads (Forster & Symons, 1987a, b). A limited number of mutagenesis experiments have confirmed the need for complementary base pairs in the helices and for specific nucleotides at several of the single-stranded positions (Sampson et al., 1987; Koizumi et al., 1988, 1989: Sheldon & Symons, 1989; Ruffner et al., 1989). Presumably the essential nucleotides could either participate in tertiary interactions, coordinate catalytically important divalent metal ions, or provide functional groups necessary for activation of the labile bond. The intent of this paper is to systematically determine the sequence requirements of the hammerhead domain by changing each of the nucleotides in the conserved or semiconserved positions of the hammerhead to each of the other nucleotides and examining their activity. The resulting data allow for the revision of the hammerhead consensus se-quence, and this revised consensus was used to search other