Evolution in vitro of an RNA enzyme with altered metal dependence.
The Tetrahymena group I ribozyme catalyses a sequence-specific phosphodiester cleavage reaction on an external RNA oligonucleotide substrate in the presence of a divalent metal cation cofactor. This reaction proceeds readily with either Mg2+ or Mn2+, but no detectable reaction has been reported when other divalent cations are used as the sole cofactor. Cations such as Ca2+, Sr2+ and Ba2+ can stabilize the correct folded conformation of the ribozyme, thereby partially alleviating the Mg2+ or Mn2+ requirement. But catalysis by the ribozyme involves coordination of either Mg2+ or Mn2+ at the active site, resulting in an overall requirement for one of these two cations. Here we use an in vitro evolution process to obtain variants of the Tetrahymena ribozyme that are capable of cleaving an RNA substrate in reaction mixtures containing Ca2+ as the divalent cation. These findings extend the range of different chemical environments available to RNA enzymes and illustrate the power of in vitro evolution in generating macromolecular catalysts with desired properties.
Lehman
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Department of Chemistry Biology, Scripps Research Institute, La Jolla, California 92037.
Joyce
G F
GF
D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D002118:Calcium / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013768:Tetrahymena / Q000201:enzymology*
D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D008274:Magnesium; D002118:Calcium