- 2D Elektrophorese (1) (remove)
- Biochemical analysis of the interaction between transfer ribonucleic acid and exportin-t (2006)
- In this thesis the ternary complex of tRNA•exportin-t•Ran•GTP was studied by biochemical and biophysical methods. Firstly, photocrosslinking was used to determine the contact sites between tRNA and exportin-t. 4-Thiouridine (s4U) was introduced into tRNAPheT.th by in vitro transcription with T7 RNA polymerase and crosslinked to exportin-t by irradiation at 312 nm. The crosslinking was Ran•GTP dependent and could be competitively inhibited by other tRNA species, showing that the crosslinking was the consequence of the formation of a tRNA•exportin-t•Ran•GTP complex. The crosslinked complex of (s4U)tRNAPheT.th-exportin-t after proteinase K digestion was incubated with a primer complementary to the 3'end of the tRNAPheT.th in the primer extension reaction. The elongation of the reverse transcriptase was forced to halt at s4Us crosslinked to peptides, namely, U55, U47, U33, U20. Among them, U47 and U55 were shown to be the major crosslinking sites. U47A mutation was introduced into tRNAPheT.th to test the role of U47. The mutant tRNAPheT.th transcript was still able to bind exportin-t, though a little weaker than normal tRNAPheT.th transcript. In contrast, s4U containing mutant tRNAPheT.th U47A exhibited a much poorer capability to crosslink exportin-t. It is concluded that U47 may be a major contact site between tRNAPheT.th and exportin-t. Secondly, the binding abilities of different tRNA species in calf liver tRNABulk to exportin-t was examined by affinity chromatography on immobilized exportin-t. With a stepwise elution of 250 mM and 500 mM KCl, tRNABulk was fractionated into 2 peaks. Therefore, Different tRNAs bound to exportin-t with different affinity. Among 7 tRNAs tested, the relative affinity to exportin-t ranked as tRNALeuCAG > tRNASerGCU, tRNALeuCAA, tRNASerUGA > tRNASerAGA, tRNALeuAAG > tRNAArgACG. To interpret the different affinity of tRNAs to exportin-t, it is proposed that exportin-t preferentially exports tRNAs that are required the stronger by the protein synthesis machine. The extent of requirement of a specific tRNA by cells is supposed as the ratio between the tRNA concentration in a cell and its codon frequency in the genome, if all tRNAs are supposed to be aminoacylated and transported to ribosome equally. It was found that among 7 tRNAs identified on the 2D urea PAGE, the theoretical estimation of 5 tRNA species upon their requirement by cell ranked the same as their relative affinity to exportin-t. Finally, atomic force microscopy was used to observe exportin-t and its interaction with tRNA in a native and undisturbed state directly. Exportin-t-esterase, immobilized to trifluoromethyl ketone (TFK) modified mica surface, showed a diameter of 15 ± 2 nm. After binding tRNA and Ran•GppNHp, the diameter of the complex somewhat increased to 16 ± 2 nm.