Substitution of Val20 by Gly in elongation factor Tu. Effects on the interaction with elongation factors Ts, aminoacyl-tRNA and ribosomes.
Substitution of V20 by G in the consensus element G18HVDHGK24 of EF-Tu (referred to as EF-TuG20) strongly influences the interaction with GDP as well as the GTPase activity [Jacquet, E. & Parmeggiani, A. (1988) EMBO J. 7, 2861-2867]. In an extension of this work we describe additional properties of the mutated factor, paying particular attention to the interaction with the macromolecular ligands. Our results show that the conformational transitions induced by the mutation strongly favor the regeneration of the active complex EF-TuG20.GTP, almost as effectively as with wild-type EF-Tu in the presence of elongation factor Ts. Addition of elongation factor Ts further enhances the rate of the GDP to GTP exchange of the mutated factor. Remarkably, EF-TuG20.GDP can support the enzymatic binding of aminoacyl-tRNA to ribosome.mRNA at low MgCl2 concentration, an effect that with wild-type EF-Tu can only occur in the presence of kirromycin. Our results show that EF-TuG20.GDP shares common features with the GTP-like conformation induced by kirromycin on wild-type EF-Tu. The ability of the ribosome to activate the EF-TuG20 center for GTP hydrolysis is strongly decreased, while the stimulation by aminoacyl-tRNA is conserved. The ribosomal activity is partially restored by addition of aminoacyl-tRNA plus poly(U), showing that codon/anticodon interaction contribute to correct the anomalous interaction between ternary complex and ribosomes. The impaired activity of EF-TuG20 in poly(Phe) synthesis is related to the degree of defective GTP hydrolysis and, most interestingly, it is characterized by a striking increase of the fidelity of translation at high MgCl2 concentration. This effect probably depends on a more selective recognition of the ternary complex by ribosome.mRNA, as a consequence of a longer pausing of EF-TuG20 on the ribosome. In conclusion, position 20 in EF-Tu is important for coordinating the allosteric mechanisms controlling the action of EF-Tu and its ligands.
Jacquet
E
E
Unité de Recherche Associée no. 240 du Centre National de al Recherche Scientifique, Ecole Polytechnique, Palaiseau, France.
Parmeggiani
A
A
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D005998:Glycine / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000378:metabolism; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000235:genetics* / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013997:Time Factors; D014633:Valine / Q000235:genetics*
D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; C023512:elongation factor Ts; D000643:Ammonium Chloride; D015636:Magnesium Chloride; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu; D014633:Valine; D005998:Glycine