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.

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer,

“…Other conserved active site interactions include an apparent 2.9 Å H-bond between the exocyclic amine of G8 and the O4 keto group of U+2, a conserved core residue ( Figure 1A) (42). The O4 atom helps align the G8 exocyclic amine such that it contacts the pro-R oxygen of G+1 ( Figure 3B).…”

The sCYMV1 hairpin ribozyme: targeting rules and cleavage of heterologous RNA

“…2,20 Kinetic determinations were performed with ribozyme concentration limiting and a large excess of substrate to determine true Michaelis constant (K M ) and k cat values. Kinetic reactions were performed in volumes 4 to 8 l with substrate RNA concentrations ranging from 60 to 4000 nm, depending on the K M of the ribozyme-substrate complex.…”

“…2,5 This structure contains the one non-canonical A:G base, however the number of Watson-Crick base pairs is 16 rather than 17 found in the sTRSV ribozyme due to one less base pair in helix 1 of the native sequence. The sCYMV1 ribozyme/substrate has 5 bp in helix 1 rather than the 6 bp found in helix 1 of the sTRSV ribozyme/substrate.…”

“…1,2 The basic hairpin ribozyme/substrate model has four helices (helices 1, 2, 3 and 4) and five loops (loops 1, 2, 3, 4, 5) named and oriented as originally described for the sTRSV hairpin ribozyme (reviewed in Ref. 3).…”

“…The minimal sequence of the native sCYMV1 hairpin ribozyme has been shown to have excellent catalytic activity and has been kinetically characterized. 2 The sTRSV hairpin ribozyme has been developed for gene therapy and successfully used to down-regulate gene expression in a number of systems. 6 The sTRSV hairpin ribozyme prefers to cleave substrates containing a BN*GUC sequence where B is any nucleotide except A and * is the site of cleavage.…”