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

Escherichia coli tRNA(SUU)(Lys), as well as human tRNA(SUU)(Lys3), has 2-th iouridine derivatives at wobble position 34 (s(2)U*(34)). Unlike the native tRNA(SUU)(Lys), the full-length, unmodified transcript of human tRNA(UUU)( Lys3) and the unmodified tRNA(UUU)(Lys3) anticodon stem/loop (ASL(UUU)(Lys3 )) did not bind AAA- or AAG-programmed ribosomes. In contrast, the complete ly unmodified yeast tRNAP(Phe) anticodon stem/loop (ASL(GAA)(Phe)) had an a ffinity (K-d = 136 +/- 49 nM) similar to that of native yeast tRNA(GmAA)(Ph e) (K-d = 103 +/- 19 nM). We have found that the single, site-specific subs titution of s(2)U(34) for U-34 to produce the modified ASL(SUU)(Lys) was su fficient to restore ribosomal binding. The modified ASL(SUU)(Lys) bound the ribosome with an affinity (K-d = 176 +/- 62 nM) comparable to that of nati ve tRNA(SUU)(Lys) (K-d = 70 +/- 7 nM). Furthermore, in binding to the ribos ome, the modified ASL(SUU)(Lys3) produced the same 16S P-site tRNA footprin t as did native E, coli tRNA(SUU)(Lys), yeast tRNA(GmAA)(Phe), and the unmo dified ASL(GAA)(Phe). The unmodified ASL(UUU)(Lys3) had no footprint at all . Investigations of thermal stability and structure monitored by UV spectro scopy and NMR showed that the dynamic conformation of the loop of modified ASL(SUU)(Lys3) was different from that of the unmodified ASL(UUU)(Lys), whe reas the stems were isomorphous. Based on these and other data, we conclude that s(2)U(34) in tRNA(SUU)(Lys) and in other s(2)u(34)-containing tRNAs i s critical for generating an anticodon conformation that leads Po effective codon interaction in all organisms. This is the first example of a single atom substitution (U-34 --> (SU34)-U-2) that confers the property of riboso mal binding on an otherwise inactive tRNA.