Anticodon Domain Methylated Nucleosides of Yeast tRNAPhe Are Significant Recognition Determinants in the Binding of a Phage Display Selected Peptide

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Abstract
The contributions of the natural modified nucleosides to RNA identity in protein/RNA interactions are not understood. We had demonstrated that 15 amino acid long peptides could be selected from a random phage display library using the criterion of binding to a modified, rather than unmodified, anticodon domain of yeast tRNAPhe (ASLPhe). Affinity and specificity of the selected peptides for the modified ASLPhe have been characterized by fluorescence spectroscopy of the peptides' tryptophans. One of the peptides selected, peptide tF2, exhibited the highest specificity and most significant affinity for ASLPhe modified with 2‘-O-methylated cytidine-32 and guanosine-34 (Cm32 and Gm34) and 5-methylated cytidine-40 (m5C40) (Kd = 1.3 ± 0.4 μM) and a doubly modified ASLPhe-Gm34,m5C40 and native yeast tRNAPhe (Kd ≅ 2.3 and 3.8 μM, respectively) in comparison to that for the unmodified ASLPhe (Kd = 70.1 ± 12.3 μM). Affinity was reduced when a modification altered the ASL loop structure, and binding was negated by modifications that disfavored hairpin formation. Peptide tF2's higher affinity for the ASLPhe-Cm32,Gm34,m5C40 hairpin and fluorescence resonance energy transfer from its tryptophan to the hypermodified wybutosine-37 in the native tRNAPhe placed the peptide across the anticodon loop and onto the 3‘-side of the stem. Inhibition of purified yeast phenylalanyl-tRNA synthetase (FRS) catalyzed aminoacylation of cognate yeast tRNAPhe corroborated the peptide's binding to the anticodon domain. The phage-selected peptide tF2 has three of the four amino acids crucial to G34 recognition by the β-structure of the anticodon-binding domain of Thermus thermophilus FRS and exhibited circular dichroism spectral properties characteristic of β-structure. Thus, modifications as simple as methylations contribute identity elements that a selected peptide specifically recognizes in binding synthetic and native tRNA and in inhibiting tRNA aminoacylation.

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