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

摘要

Escherichia coli tRNA(SUU)(Lys), as well as human tRNA(SUU)(Lys3), has 2-thiouridine 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 completely unmodified yeast tRNAP(Phe) anticodon stem/loop (ASL(GAA)(Phe)) had an affinity (K-d = 136 +/- 49 nM) similar to that of native yeast tRNA(GmAA)(Phe) (K-d = 103 +/- 19 nM). We have found that the single, site-specific substitution of s(2)U(34) for U-34 to produce the modified ASL(SUU)(Lys) was sufficient to restore ribosomal binding. The modified ASL(SUU)(Lys) bound the ribosome with an affinity (K-d = 176 +/- 62 nM) comparable to that of native tRNA(SUU)(Lys) (K-d = 70 +/- 7 nM). Furthermore, in binding to the ribosome, the modified ASL(SUU)(Lys3) produced the same 16S P-site tRNA footprint as did native E, coli tRNA(SUU)(Lys), yeast tRNA(GmAA)(Phe), and the unmodified ASL(GAA)(Phe). The unmodified ASL(UUU)(Lys3) 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(SUU)(Lys3) was different from that of the unmodified ASL(UUU)(Lys), whereas 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 is 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 ribosomal binding on an otherwise inactive tRNA. [References: 29]