Catalytic RNA reactions of yeast tRNAPhe fragments

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Abstract
We describe 12 new catalytic RNA reactions which are intermolecular variants of the well-known intramolecular Pb(2+)-promoted hydrolysis of yeast tRNA(Phe). Fragments derived from the native yeast tRNA(Phe) which possess the T stem-loop can function as catalysts for the site-specific hydrolysis at p18 of D stem-loop-containing fragments. An initial report described the catalytic cleavage of an unmodified T7 transcript corresponding to the 5' half of tRNA(Phe) by a 3' half-molecule derived from the native tRNA. [Sampson, J. R., Sullivan, F. X., Behlen, L. S., DiRenzo, A. B., & Uhlenbeck, O. C. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, 267-275]. We have investigated the trans reaction further by creating a family of substrate and catalyst RNA molecules by dissection of the native tRNA(Phe) using a combination of chemical and enzymatic methods. A search for cleavage activity in trans was conducted using a combinatorial approach with the available T and D stem-loop-containing fragments. Twelve combinations were found to be catalytic, and initial rates, kcat's, and Km's are reported for each. The kcat's for the reactions differ by approximately 20-fold, whereas Km's vary by only approximately 2-fold. Differences in some of the cleavage rates argue that tertiary interactions present in the intact molecule can be reconstituted in the fragment combinations. Secondary structural features remote from the cleavage site can also affect the apparent cleavage rates. A minimum catalytic complex consisting of a substrate fragment corresponding to nucleotides 1-24 of the native molecule and a catalytic RNA corresponding to 46-76 is identified. This complex is of interest since the transition state for cleavage involves only three helices, with no elements of the anticodon required for cleavage. This is reminiscent of the proposed secondary structure of the hammerhead catalytic RNA cleavage motif.

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