Translational suppression by hammerhead ribozymes and inactive variants inS-cerevisiae

The activity of hammerhead ribozymes in S. cerevisiae was assessed using tw o ribozymes that were designed to intramolecularly attack the hepatitis B v iral X mRNA. The ribozymes effectively suppressed the expression of the X-l acZ fusion gene, when they were inserted at the 5' end of the X mRNA. The r ibozymes cleaved the target RNA efficiently at the targeted phosphodiester bond, but the inactive mutants carrying GS-to-A substitution in the core di d not, as the total RNA preparations of yeast extracts was assayed by prime r extension. These G5A mutants, however, exerted the suppression as effecti vely as the wild-type ribozymes. The results, with several mutations introd uced to a ribozyme, suggested that either mere formation of hammerhead-like structures with the three stems, or the formation of any two stems, could inhibit translation. Thus, the hammerhead-like structures, leading to cleav age or not, could effectively suppress translation, especially when formed around the initiation codon. The GS-to-A and U7-to-G mutations and replacem ent of the stem-II hairpin tetraloop did not appear to affect the formation of the inhibitory structure(s). The inhibition that was observed when stem s I and III were directly connected without a loop or with a stem II hairpi n was completely when they were connected with only the loop of stem II (no t containing the stem portion). Published by Elsevier Science B.V.