RESUMO
GTP hydrolysis by elongation factor Tu (EF-Tu), a translational GTPase that delivers aminoacyl-tRNAs to the ribosome, plays a crucial role in decoding and translational fidelity. The basic reaction mechanism and the way the ribosome contributes to catalysis are a matter of debate. Here we use mutational analysis in combination with measurements of rate/pH profiles, kinetic solvent isotope effects, and ion dependence of GTP hydrolysis by EF-Tu off and on the ribosome to dissect the reaction mechanism. Our data suggest that--contrary to current models--the reaction in free EF-Tu follows a pathway that does not involve the critical residue H84 in the switch II region. Binding to the ribosome without a cognate codon in the A site has little effect on the GTPase mechanism. In contrast, upon cognate codon recognition, the ribosome induces a rearrangement of EF-Tu that renders GTP hydrolysis sensitive to mutations of Asp21 and His84 and insensitive to K(+) ions. We suggest that Asp21 and His84 provide a network of interactions that stabilize the positions of the γ-phosphate and the nucleophilic water, respectively, and thus play an indirect catalytic role in the GTPase mechanism on the ribosome.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação/genética , Catálise , Domínio Catalítico , Códon/genética , Códon/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Guanosina Trifosfato/química , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Mutação , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Ligação Proteica , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/químicaRESUMO
Speed and accuracy of protein synthesis are fundamental parameters for the fitness of living cells, the quality control of translation, and the evolution of ribosomes. The ribosome developed complex mechanisms that allow for a uniform recognition and selection of any cognate aminoacyl-tRNA (aa-tRNA) and discrimination against any near-cognate aa-tRNA, regardless of the nature or position of the mismatch. This review describes the principles of the selection-kinetic partitioning and induced fit-and discusses the relationship between speed and accuracy of decoding, with a focus on bacterial translation. The translational machinery apparently has evolved towards high speed of translation at the cost of fidelity.
Assuntos
Evolução Molecular , Biossíntese de Proteínas , Ribossomos/genética , Proteínas de Bactérias/química , Domínio Catalítico , Códon/química , Ativação Enzimática , Escherichia coli/química , Escherichia coli/genética , GTP Fosfo-Hidrolases/química , Guanosina Trifosfato/química , Hidrólise , Cinética , Fator Tu de Elongação de Peptídeos/química , Peptídeos/química , Aminoacil-RNA de Transferência/química , Ribossomos/química , Fatores de TempoRESUMO
Legionella pneumophila, which is the causative organism of Legionnaires disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt's), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNA(Phe) and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNA(His) and Phe-tRNA(Phe)) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt's and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt's.
Assuntos
Glucosiltransferases/metabolismo , Legionella pneumophila/enzimologia , Fator 1 de Elongação de Peptídeos/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Biocatálise , Linhagem Celular , Cromatografia Líquida , Clonagem Molecular , Glucosiltransferases/genética , Glicosilação , Macrófagos/parasitologia , Camundongos , Modelos Moleculares , Fator 1 de Elongação de Peptídeos/química , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10⻳, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in k(cat) values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near- and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation.
Assuntos
Códon/genética , Guanosina Trifosfato/metabolismo , Elongação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , RNA de Transferência Aminoácido-Específico , Aminoacil-RNA de Transferência , Ribossomos/metabolismo , Escherichia coli , CinéticaRESUMO
Infrared A radiation (IRA) is a major component of sunlight. Similar to ultraviolet (UV) B and UVA, IRA induces gene transcription. In contrast to the UV response very little is known about the IRA response. In the present study, IRA-induced expression of matrix metalloproteinase-1 (MMP-1) was found to be mediated by the formation of intracellular reactive oxygen species (ROS). Staining of IRA-irradiated cells with MitoSox revealed an increase in mitochondrial superoxide anion production and treatment of fibroblasts with the mitochondrial targeted antioxidant MitoQ completely abrogated the IRA, but not the UVB or UVA1, response. ROS relevant for IRA-induced signaling originated from the mt electron transport chain, because (i) chemical inhibition of the electron transport chain prevented IRA, but not UVB or UVA1, radiation-induced MMP-1 expression, (ii) rho0 fibroblasts specifically failed to increase MMP-1 expression in response to IRA, and (iii) peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) overexpressing fibroblasts with increased electron transport chain content were hypersensitive to IRA radiation-induced gene expression. Thus, IRA, in contrast to UV, elicits a retrograde signaling response in human skin.