Purification of aminoacyl-tRNA by affinity chromatography on immobilized Thermus thermophilus EF-Tu.GTP.
Elongation factor Tu from Thermus thermophilus containing six histidine residues on its C-terminus, EF-Tu(CHis6), was used for purification of aminoacyl-tRNA isoacceptors, from aminoacylated bulk tRNA, by affinity chromatography. Preformed aminoacyl-tRNA.EF-Tu(CHis6).GTP ternary complexes were immobilized on Ni(2+)-nitriloacetic acid agarose and the aminoacyl-tRNA was eluted at high ionic strength or with buffers containing GDP. Compared to alternative methods, the reported immobilization by C-terminal histidine residues does not lead to loss of EF-Tu's affinity for aminoacyl-tRNA. The method is well suited for preparative isolation of aminoacylated tRNA isoacceptors and as an analytical tool to test tRNA composition and aminoacylation of tRNA in crude cellular extracts.
Ribeiro
S
S
Laboratorium für Biochemie, Universität Bayreuth, Federal Republic of Germany.
Nock
S
S
Sprinzl
M
M
D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000737:chemistry*; D012346:RNA, Transfer, Amino Acyl / Q000302:isolation & purification*; D016963:Thermus thermophilus / Q000737:chemistry*
D012346:RNA, Transfer, Amino Acyl; D006639:Histidine; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu