The influence of different modifications of elongation factor Tu from Escherichia coli on ternary complex formation investigated by fluorescence spectroscopy.
A fluorescence titration assay was used to detect the effects of various modifications of E.coli elongation factor Tu on the formation of the ternary complex with aminoacyl-tRNAs. The treatment of EF-Tu.GDP with TPCK, an analogue of the 3'terminus of aminoacyl-tRNA, was found to have no influence on the conversion of EF-Tu.GDP to 'active' EF-Tu.GTP, but does decrease the affinity of the activated protein for yeast aminoacyl-tRNA by more than three orders of magnitude. Modification of the elongation factor by limited cleavage with trypsin, leading to the excision of amino acid residues 45-58, has only a minor influence on ternary complex formation. The equilibrium dissociation constant of the ternary complex with this trypsin-treated EF-Tu.GTP and E.coli Phe-tRNA(Phe) is only one order of magnitude higher than that of the ternary complex with native EF-Tu. Mutations in the amino acid residues 222 and 375 of EF-Tu also have little effect on ternary complex formation. Compared with TPCK-treated EF-Tu, the affinities of the two mutant species, designated EF-tuAR and EF-TuBO respectively, for [AEDANS-s2C]Tyr-tRNA(Tyr) are only slightly reduced and in the same range as trypsin-cleaved EF-Tu.
Ott
G
G
Laboratorium für Biochemie, Universität Bayreuth, FRG.
Jonák
J
J
Abrahams
I P
IP
Sprinzl
M
M
D004926:Escherichia coli / Q000032:analysis*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D012346:RNA, Transfer, Amino Acyl; D012360:RNA, Transfer, Phe; D012365:RNA, Transfer, Tyr; D006153:Guanosine Diphosphate; D014108:Tosylphenylalanyl Chloromethyl Ketone; D006160:Guanosine Triphosphate; D014357:Trypsin; D010444:Peptide Elongation Factor Tu