Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis

Abstract

During protein synthesis, deacylated transfer RNAs leave the ribosome via an exit (E) site after mRNA translocation. How the ribosome regulates tRNA dissociation and whether functional linkages between the aminoacyl (A) and E sites modulate the dynamics of protein synthesis have long been debated. Using single molecule fluorescence resonance energy transfer experiments, we find that, during early cycles of protein elongation, tRNAs are often held in the E site until being allosterically released when the next aminoacyl tRNA binds to the A site. This process is regulated by the length and sequence of the nascent peptide and by the conformational state, detected by tRNA proximity, prior to translocation. In later cycles, E-site tRNA dissociates spontaneously. Our results suggest that the distribution of pretranslocation tRNA states and posttranslocation pathways are correlated within each elongation cycle via communication between distant subdomains in the ribosome, but that this correlation between elongation cycle intermediates does not persist into succeeding cycles.FRET | alternating wavelength laser excitation (ALEX) | multi-translation cycles | classical state | hybrid state

Structural Implications of Universal Complementarities in Translation—High Accuracy at the Decoding Site

“…However, the experimental findings of Nierhaus and his coworkers [46]- [50] described above have recently been refused to accept by many research groups [51]- [54]. Chen et al [53] analysed how E-site tRNA is dissociated early in protein synthesis. They denoted the two options as the 2-3-2 tRNA and 2-1-2 tRNA pathways, where the integers indicate the number of tRNAs occupying the ribosome during the conversion of the POST complex to the PRE complex.…”

“…Petropoulos and Green [54] used the same buffer, and yet refused the negative cooperativity between A-and E sites [46]. Chen et al [53] observed that the 2-3-2 pathway only occurred shortly after initiation and followed by consecutive 2-1-2 pathways and that the translation pathway is mainly determined by the PRE conformational states (either classical or hybrid). They used TAM 15 buffer, which contains 15 mMol MgAc 2 [53].…”

“…Chen et al [53] observed that the 2-3-2 pathway only occurred shortly after initiation and followed by consecutive 2-1-2 pathways and that the translation pathway is mainly determined by the PRE conformational states (either classical or hybrid). They used TAM 15 buffer, which contains 15 mMol MgAc 2 [53]. It seems to me that they are using too high Mg 2+ concentration, even if they lowered it from 15 to 7 mMol [53].…”

“…They used TAM 15 buffer, which contains 15 mMol MgAc 2 [53]. It seems to me that they are using too high Mg 2+ concentration, even if they lowered it from 15 to 7 mMol [53]. Every group should discuss with each other in terms of accuracy in translation.…”

Bacterial replication, transcription and translation: mechanistic insights from single-molecule biochemical studies

“…However, the early rounds of elongation were recently examined using smFRET, and this uncovered an additional pathway of tRNA release that was not observed in later rounds of elongation 61 . In this pathway, the deacylated tRNA remains associated with the ribosomal E-site until the next charged tRNA binds to the A-site, at which point the deacylated tRNA is ejected from the E-site.…”