A study of binding features between exportin-t and Thermus thermophilus tRNA(Phe) using a photo-cross-linking method

The interaction of tRNA and its carrier, exportin-t, was studied by photoaffinity cross-linking of randomly s (4)U-substituted tRNA (Phe) T.th in vitro transcript that was able to form complex with exportin-t and ran.GTP. We found that in the ternary complex, tRNA (Phe) T.th was cross-linked only to exportin-t, not to ran.GppNHp (an analogue of ran.GTP), suggesting that tRNA had much stronger contact with exportin-t than with ran.GTP. The contact sites between tRNA (Phe) T.th and exportin-t were supposed to be the cross-linking sites in this study and further determined by primer extension analysis. The major cross-linking sites were U55, U47, U20, and U33. Apart from the footprinting data on the contact sites between exportin-t and tRNA, which are mainly on the acceptor arm and TPsiC region, our results showed that exportin-t bound tRNA in a very extensive way, and the anticodon loop and D loop might also contact, though weakly, the tRNA carrier. Using the site-directed mutagenesis method, we also found that while position 47 in tRNA (Phe) T.th is important for the binding interaction, the identity of the nucleotide at this position is not.