Smaller, faster ribozymes reveal the catalytic core of Neurospora VS RNA.
We have investigated the structural requirements for cis-cleavage of the VS ribozyme by designing deletions, substitutions, and circular permutations based on the secondary structure model. Four of the six helices predicted in the model have been shortened, resulting in self-cleaving RNAs of only 121 to 126 nucleotides. Remarkably, the shorter ribozymes exhibit a 30 to 40-fold faster cis-cleavage rate. The increase in activity results from disrupting an inhibitory helix whose 5' side contains bases upstream of the cleavage site, and from constructing a circular permutation that tethers the helix containing the cleavage site to a shortened version of the rest of the ribozyme. The non-essential regions identified by the deletions map to the periphery of a recently proposed structure model, revealing a central ribozyme core that contains the essential structural elements required for activity of the VS ribozyme.
Rastogi
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Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
Collins
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D001483:Base Sequence; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium; D008969:Molecular Sequence Data; D009491:Neurospora / Q000235:genetics*; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion
D016337:RNA, Catalytic; D012331:RNA, Fungal; D008274:Magnesium