RESUMO
Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H]12+ ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an Elab energy of 480-500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H]12+ EF-Tu ion showed similar qualitative behavior to the experimental results.
Assuntos
Antibacterianos , Simulação de Dinâmica Molecular , Fator Tu de Elongação de Peptídeos , Desdobramento de Proteína , Espectrometria de Massas por Ionização por Electrospray , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Antibacterianos/químicaRESUMO
The frequency of errors upon decoding of messenger RNA by the bacterial ribosome is low, with one misreading event per 1 × 104 codons. In the universal genetic code, the AUN codon box specifies two amino acids, isoleucine and methionine. In bacteria and archaea, decoding specificity of the AUA and AUG codons relies on the wobble avoidance strategy that requires modification of C34 in the anticodon loop of isoleucine transfer RNAIleCAU (tRNAIleCAU). Bacterial tRNAIleCAU with 2-lysylcytidine (lysidine) at the wobble position deciphers AUA while avoiding AUG. Here we report cryo-electron microscopy structures of the Escherichia coli 70S ribosome complexed with elongation factor thermo unstable (EF-Tu) and isoleucine-tRNAIleLAU in the process of decoding AUA and AUG. Lysidine in tRNAIleLAU excludes AUG by promoting the formation of an unusual Hoogsteen purine-pyrimidine nucleobase geometry at the third position of the codon, weakening the interactions with the mRNA and destabilizing the EF-Tu ternary complex. Our findings elucidate the molecular mechanism by which tRNAIleLAU specifically decodes AUA over AUG.
Assuntos
Microscopia Crioeletrônica , Escherichia coli , Modelos Moleculares , Fator Tu de Elongação de Peptídeos , RNA de Transferência de Isoleucina , Ribossomos , Fator Tu de Elongação de Peptídeos/metabolismo , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Ribossomos/química , RNA de Transferência de Isoleucina/metabolismo , RNA de Transferência de Isoleucina/química , RNA de Transferência de Isoleucina/genética , Códon/metabolismo , Códon/genética , Anticódon/química , Anticódon/metabolismo , Conformação de Ácido Nucleico , Isoleucina/metabolismo , Isoleucina/química , RNA Mensageiro/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , Lisina/análogos & derivados , Nucleosídeos de PirimidinaRESUMO
To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3-6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.
Assuntos
Proteínas de Bactérias , Resposta ao Choque Frio , Fatores de Terminação de Peptídeos , Biossíntese de Proteínas , Psychrobacter , Proteínas Ribossômicas , Ribossomos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Fator Tu de Elongação de Peptídeos/ultraestrutura , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Ribossomos/química , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Psychrobacter/química , Psychrobacter/genética , Psychrobacter/metabolismo , Psychrobacter/ultraestrutura , Microscopia Crioeletrônica , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/ultraestruturaRESUMO
Targeting translational factor proteins (TFPs) presents significant promise for the development of innovative antitubercular drugs. Previous insights from antibiotic binding mechanisms and recently solved 3D crystal structures of Mycobacterium tuberculosis (Mtb) elongation factor thermo unstable-GDP (EF-Tu-GDP), elongation factor thermo stable-EF-Tu (EF-Ts-EF-Tu), and elongation factor G-GDP (EF-G-GDP) have opened up new avenues for the design and development of potent antituberculosis (anti-TB) therapies.