RESUMO
Initiation factor (IF) 2 controls the fidelity of translation initiation by selectively increasing the rate of 50S ribosomal subunit joining to 30S initiation complexes (ICs) that carry an N-formyl-methionyl-tRNA (fMet-tRNAfMet). Previous studies suggest that rapid 50S subunit joining involves a GTP- and fMet-tRNAfMet-dependent "activation" of IF2, but a lack of data on the structure and conformational dynamics of 30S IC-bound IF2 has precluded a mechanistic understanding of this process. Here, using an IF2-tRNA single-molecule fluorescence resonance energy transfer signal, we directly observe the conformational switch that is associated with IF2 activation within 30S ICs that lack IF3. Based on these results, we propose a model of IF2 activation that reveals how GTP, fMet-tRNAfMet, and specific structural elements of IF2 drive and regulate this conformational switch. Notably, we find that domain III of IF2 plays a pivotal, allosteric, role in IF2 activation, suggesting that this domain can be targeted for the development of novel antibiotics.
Assuntos
Escherichia coli/fisiologia , Fator de Iniciação 2 em Procariotos/fisiologia , Biossíntese de Proteínas/fisiologia , Subunidades Ribossômicas Maiores de Bactérias/fisiologia , Subunidades Ribossômicas Menores de Bactérias/fisiologia , Regulação Alostérica/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Transferência Ressonante de Energia de Fluorescência/métodos , Guanosina Trifosfato/metabolismo , Cinética , Modelos Biológicos , Mutação , Fator de Iniciação 2 em Procariotos/química , Conformação Proteica , Domínios Proteicos/fisiologia , RNA de Transferência de Metionina/metabolismo , Imagem Individual de Molécula/métodosRESUMO
A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.
