Studies of the complex between transfer RNAs with complementary anticodons: I. Origins of enhanced affinity between complementary triplets

Abstract

We used the temperature-jump method to study the complex between yeast t RNA Pheand Escherichia coli tRNA Glu, which have the complementary anticodons GmAA and s 2UUC, respectively. The binding constant (3.6 × 10 5 m −1 at 25 °C) is about six orders of magnitude larger than expected for two complementary trinucleotides. The association rate constant (3 × 10 6 m −1 at 25 °C) is similar to typical values observed for oligonucleotides, so the enhanced affinity in the tRNA · tRNA complex is due entirely to a much slower dissociation than expected for a three base-pair helix. We found an association enthalpy of −25 kcal/mol, nearly twice as large as expected for two stacking interactions in a three base-pair helix. The association entropy (−58 cal/deg per mol) is close to the expected value. The reaction occurs with a single relaxation, and therefore does not involve any slow reorganization of the tRNA molecule. We studied structural variations to investigate the origin of affinity enhancement. The following general factors are important. (1) The “loop constraint”, or closure of the two anticodon sequences into hairpin loops, accounts for about a factor 50 in the affinity. (2) “Dangling ends”, or non-complementary nucleotides at the end of the double helix contribute strongly to the affinity. (3) Modified nucleotides, like the Y base, in the dangling ends can contribute a special stabilization of up to a factor seven. These observations can be understood in terms of a model in which the short three base-pair helix is sandwiched between stacked bases and hence stabilized. The potential importance of loop-loop interactions and stacking effects for codon-anticodon bonding is emphasized. The results suggest a possible simple physical basis for the evolutionary choice of a triplet coding system.