Transcription reaction was carried out in the presence of Biotin-GMP to generate 5 biotinylated RNA. smFRET experiments Detailed methods regarding purification of ribosomal subunits, translation factors, tRNA, and specific reagent concentrations, buffer composition, and incubation times have been previously described30,32,33. of dissociation and non-productive rebinding. The glycyl amino-acid moiety on the A-site Gly-tRNA manages to overcome the arrest by CHL. Our results illuminate the mechanism of CHL and LZD action through their interactions with the ribosome, the nascent peptide and the incoming amino acid, perturbing elongation dynamics. During translation, the ribosome catalyzes peptide bond formation between chemically and structurally diverse substrates. To accomplish this task, the ribosome precisely positions the peptidyl-tRNA (pept-tRNA) in the P site and aminoacyl-tRNA (aa-tRNA) in the A site1. The formation of the new peptide bond sAJM589 results in the transfer of the nascent peptide from the P- to the A-site tRNA, extending the nascent peptide by a single Slc7a7 amino acid. Upon peptidyl transfer, the 50S subunit rotates by sAJM589 7?10 relative to the 30S subunit2C6 and tRNAs assume the hybrid state, with their anticodons remaining in the P and A sites in the small subunit, but their acceptor ends moved to the P and E sites in the large subunit (P/E and A/P tRNA states)4,7. A subsequent translocation step moves the mRNA and tRNA anticodon stem-loops to the E and P sites with the ribosome ratcheting back to the non-rotated state. Various intermediate rotation8C10 and small-subunit conformational11 states are sampled during these transitions, although some of them might be too short-lived to be experimentally identified. Many protein synthesis inhibitors act by sterically disrupting the association and/or positioning of the substrates in the PTC and thus, blocking peptide bond formation12C15. Two such small molecules are the antibiotics chloramphenicol (CHL) and linezolid (LZD) (Fig 1a). CHL is a long-known antibiotic initially isolated from ribosomal small subunits at helix 44 sAJM589 with Cy3B (Cy3B-30S) and labeled large subunits at helix 101 with the quencher BHQ-2 (BHQ-50S)27,29. The one-color FRET signal between the dyes allows the monitoring of ribosomal conformation changes during translation (Fig 2a, green trace). Before aa-tRNA binding, the ribosome assumes a non-rotated state, characterized by substantially quenched Cy3B fluorescence state because of its proximity to BHQ-2 on the large ribosomal subunit. Upon the aa-tRNA accommodation, the peptidyl-transfer reaction induces a transition to the rotated state, detected as a higher Cy3B intensity due to the increased distance between Cy3B and BHQ-2. Subsequently, translocation of the ribosome to the next codon resets the non-rotated state with the deacylated tRNA rapidly departing from the E site30, completing one cycle of translation elongation. To increase further the accuracy of the assignment of translation cycles based on monitoring the intersubunit rotation, we used the binding of fluorescently-labeled tRNAPhe labeled with Cy5 (at the naturally modified acp3U47 residue31; see Methods) at F2 and sAJM589 F5 codons28 (Fig 2a, red trace). The tRNA signal appears when aa-tRNA binds to the A site of the ribosome and persists after its sAJM589 translocation to the P site until the subsequent cycle of elongation places labeled tRNA in the E site for dissociation. Open in a separate window Figure 2. Monitoring the drug-induced translation arrest using smFRET-based assay.a. Top: Diagram of smFRET-based assay to monitor ribosome structural changes coupled to translation elongation progression. Using Cy3BCBHQ-2 dye-quencher pair, non-rotated and rotated ribosome conformations are tracked. Binding of labeled specific tRNA (Phe-(Cy5)-tRNAPhe) is used to enhance fidelity of state transitions. Bottom: diagram of expected fluorescence intensities matching the structural states depicted immediately above. b. Representative traces for experiments without any antibiotics (similar results observed for = 87, 100 and 139 for conditions no drug, 1M CHL and 5M LZD, respectively). c. Processivity of translation over the first six codons within the mRNA open reading frame at different conditions, measured as percentage of ribosomes that translated a particular codon over the entire population. d. Measurement of Cy5 pulse-durations from Phe-(Cy5)-tRNAPhe binding events at Phe codon 2 (F2) and Phe codon 5 (F5) for different conditions. Error bars represent 95% confidence interval from fitting the single-exponential distribution. Sample sizes for each conditions are identical in 2b-d. Fluorescence intensity states corresponding to the non-rotated and rotated state for translating the first five codons were assigned as follows: a decrease in the Cy3B fluorescence intensity followed by a concurrent increase of both Cy3B and Cy5 intensities were assigned as a translation initiation event (binding of BHQ-50S to the surface-tethered mRNACCy3B-30S pre-initiation complex) and transition from non-rotated to rotated state for decoding of the first Phe (F2) codon and first peptide bond formation, respectively (Fig 2a,?,b).b). Observed non-rotated and rotated intensity levels observed during this translation of the first F2 codon were used to assign subsequent Cy3B fluorescence intensity changes between the non-rotated and rotated states for the first five codons (F2-K3-A4-F5-K6) following the start codon M1 in the MFKAFK mRNA construct. Concurrent decreases of the.