Identification of separate structural features that affect rate and cation concentration dependence of self-cleavage by the Neurospora VS ribozyme.
The cleavage site of the Neurospora VS ribozyme is located in an internal loop in a hairpin called stem-loop I. Stem-loop I undergoes a cation-dependent structural change to adopt a conformation, termed shifted, that is required for activity. Using site-directed mutagenesis and kinetic analyses, we show here that the insertion of a single-stranded linker between stem-loop I and the rest of the ribozyme increases the observed self-cleavage rate constant by 2 orders of magnitude without affecting the Mg(2+) requirement of the reaction. A distinct set of mutations that favors the formation of the shifted conformation of stem-loop I decreases the Mg(2+) requirement by an order of magnitude with little or no effect on the observed cleavage rate under standard reaction conditions. Similar trends were seen in reactions that contained Li(+) instead of Mg(2+). Mutants with lower ionic requirements also exhibited increased thermostability, providing evidence that the shifted conformation of stem-loop I favors the formation of the active conformation of the RNA. In natural, multimeric VS RNA, where a given ribozyme core is flanked by one copy of stem-loop I immediately upstream and another copy 0.7 kb downstream, cleavage at the downstream site is strongly preferred, providing evidence that separation of stem-loop I from the ribozyme core reflects the naturally evolved organization of the RNA.
Poon
Alan H L
AH
Department of Molecular and Medical Genetics, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada.
Olive
Joan E
JE
McLaren
Meredith
M
Collins
Richard A
RA
D001483:Base Sequence; D002412:Cations; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009491:Neurospora / Q000201:enzymology*; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002412:Cations; D016337:RNA, Catalytic; D008274:Magnesium