Catalytic Properties of Hairpin Ribozymes Derived from Chicory Yellow Mottle Virus and Arabis Mosaic Virus Satellite RNAs

Abstract

Regions of the negative strands of the satellite RNAs of chicory yellow mottle virus (sCYMV1) and arabis mosaic virus (sArMV) have similarity in sequence and predicted secondary structure compared to the tobacco ringspot virus satellite RNA (sTRSV) hairpin ribozyme, suggesting that they may also be catalytic RNAs of a similar type. Our experiments show that the hairpin ribozyme-like sequences derived from sCYMV1 and sArMV have high phosphodiesterase activity. The Kcat values determined are similar to that of the highly active native sTRSV hairpin ribozyme under the same conditions, although the Km values are much higher. The Km of the sArMV ribozyme was reduced 3-fold, with no change in kcat, by extending substrate hybridization in helix 2. Additionally, the three hairpin ribozymes prefer different GUX sequences on the immediate 3'-side of the cleavage site. The sTRSV hairpin ribozyme cleaves GUX substrates with catalytic efficiencies in the relative order GUC > GUU > GUG = GUA. The sCYMV1 ribozyme cleaves GUA > GUC, GUG, GUU. The sArMV ribozyme prefers GUA > GUG > GUU > GUC. The functional domain, regulating substrate selection at this position, must reside in the nucleotides that vary between the ribozyme--substrate complexes. The sTRSV ribozyme is most efficient at cleaving GUC complexes, while the sCYMV1 and sArMV ribozymes are most efficient for cleaving GUA-containing sequences.