Site-directed mutagenesis of elongation factor Tu. The functional and structural role of residue Cys81.
A Cys residue located in the second consensus sequence element (DCPG) of the GTP-binding region is highly conserved in bacterial elongation factors (EF) Tu. Chemical modification of this Cys81 in EF-Tu from Escherichia coli by N-tosyl-L-phenylalanine chloromethane [Jonák, J., Petersen, T. E., Clark, B. F. C. & Rychlík, I. (1982) FEBS Lett. 150, 485-488], and of homologous Cys residues in other bacterial EF-Tu, selectively blocks the binding of Xaa-tRNA. We have substituted Cys81 with Gly using site-directed mutagenesis of the EF-Tu-encoding tuf A gene. This substitution induces a partial inhibition (20-70%) of: (a) poly(U)-directed poly(Phe) synthesis; (b) EF-Tu/Xaa-tRNA interaction, determined as protection by EF-Tu of the non-enzymic deacylation of Xaa-tRNA; (c) EF-Tu-dependent binding of Xaa-tRNA to the mRNA/ribosome complex and (d) the intrinsic GTPase reaction, that is also less sensitive to stimulation by Xaa-tRNA. Our results thus provide evidence that Cys81, though important, is not essential for the binding of Xaa-tRNA to EF-Tu. The accuracy in poly(Phe) synthesis, measured as misincorporation of Leu, was increased. Both the binding affinity of [C81G]EF-Tu for the nucleotide and the resistance against thermal denaturation are more strongly decreased in the case of the GDP-bound state than in the case of the GTP-bound state, suggesting that Cys81 plays a more specific role in the former conformation. The sensitivity to N-tosyl-L-phenylalanine chloromethane is decreased by 80% but not totally lost. The inhibition by N-tosyl-L-phenylalanine chloromethane treatment of the function of EF-Tu appears to be a consequence of steric hindrance and/or of an altered conformation of EF-Tu.GTP. The lower activities of [C81G]EF-Tu are probably due to long-range effects, mediated by an overall destabilization of the molecule that is particularly pronounced for the GDP-bound state.
Anborgh
P H
PH
Structure Diverse d'Interventions 61840 du Centre National de Recherche Scientifique, Laboratoire de Biochimie, Ecole Polytechnique, Palaiseau, France.
Parmeggiani
A
A
Jonák
J
J
D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010452:Peptide Biosynthesis; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics*; D010455:Peptides; D011487:Protein Conformation; D011489:Protein Denaturation; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D013329:Structure-Activity Relationship
D010455:Peptides; D012346:RNA, Transfer, Amino Acyl; D006153:Guanosine Diphosphate; C012001:polyphenylalanine; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; D003545:Cysteine