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.
Li
Sheng
S
Department of Biochemistry, Dalian Medical University, China. lisheng1996@hotmail.com
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D028884:Karyopherins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000528:radiation effects; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*; D013891:Thiouridine / Q000737:chemistry; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014529:Uridine / Q000737:chemistry
D001426:Bacterial Proteins; D028884:Karyopherins; D012329:RNA, Bacterial; D013891:Thiouridine; D012343:RNA, Transfer; D014529:Uridine