Single atom modification (O → S) of tRNA confers ribosome binding

Abstract

Escherichia coli tRNA^Lys_SUU, as well as human tRNA^Lys3_SUU, has 2-thiouridine derivatives at wobble position 34 (s²U* ₃₄). Unlike the native tRNA^Lys_SUU, the full-length, unmodified transcript of human tRNA^Lys3_UUU and the unmodified tRNA^Lys3_UUU anticodon stem/loop (ASL^Lys3_UUU) did not bind AAA- or AAG-programmed ribosomes. In contrast, the completely unmodified yeast tRNA^Phe anticodon stem/loop (ASL^Phe_GAA) had an affinity (K_d = 136 ± 49 nM) similar to that of native yeast tRNA^Phe_GmAA (K_d = 103 ± 19 nM). We have found that the single, site-specific substitution of s²U₃₄ for U₃₄ to produce the modified ASL^Lys_SUU was sufficient to restore ribosomal binding. The modified ASL^Lys_SUU bound the ribosome with an affinity (K_d = 176 ± 62 nM) comparable to that of native tRNA^Lys_SUU (K_d = 70 ± 7 nM). Furthermore, in binding to the ribosome, the modified ASL^Lys3_SUU produced the same 16S P-site tRNA footprint as did native E. coli tRNA^Lys_SUU, yeast tRNA^Phe_GmAA, and the unmodified ASL^Phe_GAA. The unmodified ASL^Lys3_UUU had no footprint at all. Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASL^Lys3_SUU was different from that of the unmodified ASL^Lys_UUU, whereas the stems were isomorphous. Based on these and other data, we conclude that s²U₃₄ in tRNA^Lys_SUU and in other s²U₃₄-containing tRNAs is critical for generating an anticodon conformation that leads to effective codon interaction in all organisms. This is the first example of a single atom substitution (U₃₄ → s²U₃₄) that confers the property of ribosomal binding on an otherwise inactive tRNA.