MODELING ACTIVE RNA STRUCTURES USING THE INTERSECTION OF CONFORMATIONAL SPACE - APPLICATION TO THE LEAD-ACTIVATED RIBOZYME

The Pb2+ cleavage of a specific phosphodiester bond in yeast tRNA(Phe) is the classical model of metal-assisted RNA catalysis, In vitro sele ction experiments have identified a tRNA(Phe) variant, the leadzyme, t hat is very active in cleavage by Pb2+. We present here a three-dimens ional modeling protocol that was used to propose a structure for this ribozyme, and is based on the computation of the intersection of confo rmational space of sequence variants and the use of chemical modificat ion data, Sequence and secondary structure data were used in a first r ound of computer modeling that allowed identification of conformations compatible with all known leadzyme variants. Common conformations wer e then tested experimentally by evaluating the activity of analogues c ontaining modified nucleotides in the catalytic core. These experiment s led to a new structural hypothesis that was tested in a second round of computer modeling. The resulting proposal for the active conformat ion of the leadzyme is consistent with all known structural data. The final model suggests an in-line SN2 attack mechanism and predicts two Pb2+ binding sites. The protocol presented here is generally applicabl e in modeling RNAs whenever the catalytic or binding activity of struc tural analogues is known.