Following the intersubunit conformation of the ribosome during translation in real time

We report the direct observation of conformational rearrangements of the ribosome during multiple rounds of elongation. Using single-molecule fluorescence resonance energy transfer, we monitor the intersubunit conformation of the ribosome – in real time – as it proceeds from codon to codon. During each elongation cycle, the ribosome unlocks upon peptide bond formation, followed by reversion to the locked state upon translocation onto the next codon. Our data reveal both the specific and cumulative effects of antibiotics on individual steps of translation, and uncover the processivity of the ribosome as it elongates. Our approach interrogates the precise molecular events occurring at each codon of the mRNA within the full context of ongoing translation.