Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

Abstract

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein

“…All atom Gō-like structure-based simulations were performed as previously described [ 48 , 66 ]. AMBER minimized structures of the apo LIV-BP SS protein or bound to Leu, Ile, or Val (excluding hydrogens) were used as starting structures for structure-based models.…”

“…Single-molecule Förster Resonance Energy Transfer (smFRET), an experimental technique used to study the dynamics of a wide variety of biomolecules, from polymerases to ribosomes, to G-protein coupled receptors, could benefit greatly from quantitative MD simulation comparison [47][48][49][50][51]. Investigations involving MD interpretations of smFRET experiments have been a major focus for some time [52].…”

Structure of the bacterial ribosome at 2 Å resolution

“…These molecular interactions are accompanied by a wide range of conformational dynamics that contribute to translation accuracy and speed (Munro et al, 2009); (Javed and Orlova, 2019;Loveland et al, 2020;Morse et al, 2020). Because of the ribosome's essential role in supporting life, it is naturally the target of a plurality of antibiotics with diverse mechanisms of action (Arenz and Wilson, 2016).…”

“…To carry out the highly coordinated process of translation, the ribosome orchestrates the binding and readout of messenger RNA (mRNA) and transfer RNAs (tRNAs), coupled with a multitude of interactions between the small and large ribosomal subunits and a host of translation factors. These molecular interactions are accompanied by a wide range of conformational dynamics that contribute to translation accuracy and speed ( Munro et al, 2009 ; Javed and Orlova, 2019 ; Loveland et al, 2020 ; Morse et al, 2020 ). Because of the ribosome’s essential role in supporting life, it is naturally the target of a plurality of antibiotics with diverse mechanisms of action ( Arenz and Wilson, 2016 ).…”

Long-term effects of the proline-rich antimicrobial peptide Oncocin112 on the Escherichia coli translation machinery

“…We would also like to draw some similarity with a recent study from the Blanchard lab (50). They have shown using a three-color FRET study that conformational changes occurring during the accommodation step for mostly cognate tRNAs is important for EF-Tu to leave the ribosomes.…”