Site-Directed Mutagenesis of Thermus thermophilus Elongation Factor Tu. Replacement of His85, Asp81 and Arg300
| Autor: | Vladimir I. Katunin, Christian Egle, Mathias Sprinzl, Roland Kreutzer, Waltraud Zeidler, Wolfgang Wintermeyer, Marina V. Rodnina, Sofia Ribeiro, Annett Wagner |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
GTP'
Stereochemistry Molecular Sequence Data Gene Expression GTPase Peptide Elongation Factor Tu RNA Transfer Amino Acyl Protein degradation Arginine Biochemistry Structure-Activity Relationship GTP Phosphohydrolase-Linked Elongation Factors Escherichia coli Histidine Nucleotide Amino Acid Sequence Site-directed mutagenesis DNA Primers Alanine chemistry.chemical_classification Aspartic Acid Base Sequence biology Chemistry Hydrolysis Thermus thermophilus biology.organism_classification Protein Structure Tertiary Oligodeoxyribonucleotides Mutagenesis Site-Directed Isoleucine EF-Tu |
| Zdroj: | European Journal of Biochemistry. 229:596-604 |
| ISSN: | 1432-1033 0014-2956 |
| DOI: | 10.1111/j.1432-1033.1995.tb20503.x |
| Popis: | His85 in Thermus thermophilus elongation factor Tu (EF-Tu) was replaced by glutamine, leucine and glycine residues, leading to [H85Q]EF-Tu, [H85L] EF-Tu and [H85G]EF-Tu, respectively. Asp81 was replaced by alanine leading to [D81A]EF-Tu, and replacement of Arg300 provided [R300I]EF-Tu. Glycine in position 85 of domain I induces a protease-sensitive site in domain II and causes complete protein degradation in vivo. A similar effect was observed when Asp81 was replaced by alanine or Arg300 by isoleucine. Degradation is probably due to disturbed interactions between the domains of EF-Tu · GTP, inducing a protease-sensitive cleavage site in domain II. [H85Q]EF-TU, which can be effectively overproduced in Escherichia coli, is slower in poly(U)-dependent poly(Phe) synthesis, has lower affinity to aminoacyl-tRNA but shows only a slightly reduced rate of intrinsic GTP hydrolysis compared to the native protein. The GTPase of this protein variant is not efficiently stimulated by aminoacyl-tRNA and ribosomes. The slow GTPase of [H85Q]EF-Tu increases the fidelity of translation as measured by leucine incorporation into poly(Phe) in in vitro poly(U)-dependent ribosomal translation. Replacement of His85 in T. thermophilus EF-Tu by leucine completely deactivates the GTPase activity but does not substantially influence the aminoacyl-tRNA binding. [H85L]EF-Tu is inactive in poly(U)-dependent poly(Phe)-synthesis. The rate of nucleotide dissociation is highest for [H85L]EF-Tu, followed by [H85Q]EF-Tu and native T. thermophilus EF-Tu. Mutation of His85, a residue which is not directly involved in the nucleotide binding, thus influences the interaction of EF-Tu domains, nucleotide binding and the efficiency and rate of GTPase activity. |
| Databáze: | OpenAIRE |
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