Limited proteolysis and amino acid replacements in the effector region of Thermus thermophilus elongation factor Tu.
The effector region of the elongation factor Tu (EF-Tu) from Thermus thermophilus was modified by limited proteolysis or via site-directed mutagenesis. The biochemical properties of the obtained EF-Tu variants were investigated with respect to partial reactions of the functional cycle of EF-Tu. EF-Tu that was cleaved at the Arg59-Gly60 peptide bond [EF-Tu-(1-59)/EF-Tu-(60-405)] bound GDP, EF-Ts and aminoacyl-tRNA, had normal intrinsic GTPase activity and was active in poly(U)-dependent poly(Phe) synthesis. However, the GTPase activity of EF-Tu-(1-59)/EF-Tu-(60-405) was not stimulated by T. thermophilus 70S ribosomes, and its GTP-dissociation rate was increased compared with that of intact EF-Tu. EF-Tu cleaved at the Lys52-Ala53 peptide bond has properties similar to EF-Tu-(1-59)/EF-Tu-(60-405). By means of site-directed mutagenesis, Glu55 was replaced by Leu, Glu56 by Ala and Arg59 by Thr in T. thermophilus EF-Tu. These amino acid substitutions did not substantially affect either the affinity of EF-Tu. GTP for aminoacyl-tRNA or the interactions with GDP, GTP or EF-Ts. Similarly the intrinsic GTPase activity is not influenced. Replacement of Glu56 by Ala led to strong reduction in the ribosome-induced GTPase activity. This effect is specific since replacement of the neighbouring Glu55 by Leu did not affect the ribosome-induced GTPase activity. The results demonstrate that the structure of the effector region of EF-Tu in the vicinity of Arg59 is important for the control of the GTPase activity by ribosomes.
Zeidler
W
W
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Schirmer
N K
NK
Egle
C
C
Ribeiro
S
S
Kreutzer
R
R
Sprinzl
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases; D004926:Escherichia coli; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism*; D014644:Genetic Variation; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010452:Peptide Biosynthesis; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D010455:Peptides; D017354:Point Mutation; D011072:Poly U / Q000378:metabolism; D017433:Protein Structure, Secondary; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D016963:Thermus thermophilus / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D010455:Peptides; D012360:RNA, Transfer, Phe; D012365:RNA, Transfer, Tyr; D006153:Guanosine Diphosphate; D011072:Poly U; C012001:polyphenylalanine; D010450:Endopeptidases; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu