Anticodon domain methylated nucleosides of yeast tRNA(Phe) are significantrecognition determinants in the binding of a phage display selected peptide

The contributions of the natural modified nucleosides to RNA identity in pr otein/RNA interactions are not understood. We had demonstrated that 15 amin o acid long peptides could be selected from a random phage display library using the criterion of binding to a modified, rather than unmodified, antic odon domain of yeast tRNA(Phe) (ASL(Phe)). Affinity and specificity of the selected peptides for the modified ASL(Phe) have been characterized by fluo rescence spectroscopy of the peptides' tryptophans. One of the peptides sel ected, peptide t(F)2, exhibited the highest specificity and most significan t affinity for ASLPhe modified with 2'-O-methylated cytidine-32 and guanosi ne-34 (Cm-32 and Gm(34)) and 5-methylated cytidine-40 (m(5)C(40)) (K-d = 1. 3 +/- 0.4 muM) and a doubly modified ASL(Phe)-Gm(34),m(5)C(40) and native y east tRNA(Phe) (Kd congruent to 2.3 and 3.8 muM, respectively) in compariso n to that for the unmodified ASL(Phe) (K-d = 70.1 +/- 12.3 muM). Affinity w as reduced when a modification altered the ASL loop structure, and binding was negated by modifications that disfavored hairpin formation. Peptide t(F )2's higher affinity for the ASL(Phe)-Cm-32,Gm(34),m(5)C(40) hairpin and fl uorescence resonance energy transfer from its tryptophan to the hypermodifi ed wybutosine-37 in the native tRNAPhe placed the peptide across the antico don loop and onto the 3'-side of the stem. Inhibition of purified yeast phe nylalanyl-tRNA synthetase (FRS) catalyzed aminoacylation of cognate yeast t RNA(Phe) corroborated the peptide's binding to the anticodon domain. The ph age-selected peptide tF2 has three of the four amino acids crucial to G(34) recognition by the beta -structure of the anticodon-binding domain of Ther mus thermophilus FRS and exhibited circular dichroism spectral properties c haracteristic of beta -structure. Thus, modifications as simple as methylat ions contribute identity elements that a selected peptide specifically reco gnizes in binding synthetic and native tRNA and in inhibiting tRNA aminoacy lation.