RESUMO
Translational bypassing is a recoding event during which ribosomes slide over a noncoding region of the messenger RNA (mRNA) to synthesize one protein from two discontinuous reading frames. Structures in the mRNA orchestrate forward movement of the ribosome, but what causes ribosomes to start sliding remains unclear. Here, we show that elongation factor G (EF-G) triggers ribosome take-off by a pseudotranslocation event using a small mRNA stem-loop as an A-site transfer RNA mimic and requires hydrolysis of about two molecules of guanosine 5'-triphosphate per nucleotide of the noncoding gap. Bypassing ribosomes adopt a hyper-rotated conformation, also observed with ribosomes stalled by the SecM sequence, suggesting common ribosome dynamics during translation stalling. Our results demonstrate a new function of EF-G in promoting ribosome sliding along the mRNA, in contrast to codon-wise ribosome movement during canonical translation, and suggest a mechanism by which ribosomes could traverse untranslated parts of mRNAs.
Assuntos
Fator G para Elongação de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Fungos/metabolismo , Guanosina Trifosfato/metabolismo , Mutagênese Sítio-Dirigida , Fator G para Elongação de Peptídeos/genética , Biossíntese de Proteínas , RNA Mensageiro/química , RNA de Transferência/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Regiões não TraduzidasRESUMO
Protein synthesis in the cell is performed on ribosomes, large ribonucleoprotein particles, which in bacteria consist of three RNA molecules and over 50 proteins. This review summarizes recent progress in understanding the mechanisms of the elongation phase of protein synthesis. Results from rapid kinetic analysis of elongation reactions are discussed in the light of recent structural data.
Assuntos
Substâncias Macromoleculares/química , Ribossomos/química , Animais , Sítios de Ligação , Códon , Entropia , Hidrólise , Cinética , Modelos Biológicos , Peptídeos/química , Conformação Proteica , Transporte Proteico , Proteínas/química , RNA de Transferência/química , Ribossomos/metabolismoRESUMO
During the translocation step of the elongation cycle of peptide synthesis two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. Translocation is catalyzed by the elongation factor G (EF-G) and requires GTP hydrolysis. The fundamental biochemical features of the process were worked out in the 1970-80s, to a large part by A.S. Spirin and his colleagues. Recent results from pre-steady-state kinetic analysis and cryoelectron microscopy suggest that translocation is a multistep dynamic process that entails large-scale structural rearrangements of both ribosome and EF-G. Kinetic and thermodynamic data, together with the structural information on the conformational changes of the ribosome and of EF-G, provide a detailed mechanistic model of translocation and suggest a mechanism of translocation catalysis by EF-G.
Assuntos
Elongação Traducional da Cadeia Peptídica , RNA de Transferência/genética , Ribossomos/genética , Animais , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismoRESUMO
Elongation factor G (EF-G) from Escherichia coli is a large, five-domain GTPase that promotes tRNA translocation on the ribosome. Full activity requires GTP hydrolysis, suggesting that a conformational change of the factor is important for function. To restrict the intramolecular mobility, two cysteine residues were engineered into domains 1 and 5 of EF-G that spontaneously formed a disulfide cross-link. Cross-linked EF-G retained GTPase activity on the ribosome, whereas it was inactive in translocation as well as in turnover. Both activities were restored when the cross-link was reversed by reduction. These results strongly argue against a GTPase switch-type model of EF-G function and demonstrate that conformational mobility is an absolute requirement for EF-G function on the ribosome.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Ribossomos/metabolismo , Substituição de Aminoácidos , Reagentes de Ligações Cruzadas , Cisteína , Escherichia coli/metabolismo , Guanosina Difosfato/metabolismo , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Thermus thermophilus/metabolismoRESUMO
The elongation factors (EF) Tu and G and initiation factor 2 (IF2) from bacteria are multidomain GTPases with essential functions in the elongation and initiation phases of translation. They bind to the same site on the ribosome where their low intrinsic GTPase activities are strongly stimulated. The factors differ fundamentally from each other, and from the majority of GTPases, in the mechanisms of GTPase control, the timing of Pi release, and the functional role of GTP hydrolysis. EF-Tu x GTP forms a ternary complex with aminoacyl-tRNA, which binds to the ribosome. Only when a matching codon is recognized, the GTPase of EF-Tu is stimulated, rapid GTP hydrolysis and Pi release take place, EF-Tu rearranges to the GDP form, and aminoacyl-tRNA is released into the peptidyltransferase center. In contrast, EF-G hydrolyzes GTP immediately upon binding to the ribosome, stimulated by ribosomal protein L7/12. Subsequent translocation is driven by the slow dissociation of Pi, suggesting a mechano-chemical function of EF-G. Accordingly, different conformations of EF-G on the ribosome are revealed by cryo-electron microscopy. GTP hydrolysis by IF2 is triggered upon formation of the 70S initiation complex, and the dissociation of Pi and/or IF2 follows a rearrangement of the ribosome into the elongation-competent state.
