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

Abstract

We report the direct observation of conformational rearrangements of the ribosome during multiple rounds of elongation. Using single-molecule fluorescence resonance energy transfer, we monitored 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, then reverts 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.

Figure 1. Intersubunit FRET monitors the elongation cycle at each codon during translation

(a) Single-molecule translation assay. Cy5-labeled 50S subunits, ternary complex, and EF-G are delivered to surface-immobilized single PICs containing Cy3-labeled 30S subunits and illuminated at 532 nm; both Cy3 and Cy5 fluorescence are simultaneously detected. (b) Immobilization with an mRNA coding for 6 phenylalanines (6F) permits the observation of ribosome conformation during multiple rounds of elongation via the intersubunit FRET signal. The arrival of FRET corresponds to 50S subunit joining during initiation, and is followed by multiple cycles of high-low-high FRET, each reporting on ribosome unlocking and locking during one round of elongation.

Figure 2. The number of complete FRET cycles is controlled by coding length

Histograms of the number of complete FRET cycles observed for single ribosomes on mRNAs coding 3 (3F), 6 (6F), and 12 (12F) phenylalanines, as well as a heteropolymeric mRNA coding alternating phenylalanine and lysine residues (6FK) (139 ribosomes, 303 ribosomes, 1034, and 1145 ribosomes, respectively). Histograms are normalized to the number of ribosomes showing one FRET cycle.

Figure 3. Each FRET cycle reports on one round of elongation at a distinct codon

(a) Observing intersubunit FRET on a three codon mRNA coding phenylalanine-lysine-phenylalanine (FKF) allows control over the number of codons translated, by exclusion or inclusion of Lys-tRNA^Lys ternary complex in the delivery mixture. (b) Histograms of the number of complete FRET cycles observed for single ribosomes on the FKF mRNA in either the absence (137 ribosomes) or presence (333 ribosomes) of Lys-tRNA^Lys ternary complex; histograms are normalized to the number of molecules showing one FRET cycle. (c) Normalized 2D histograms of FRET and Phe-(Cy2)tRNA^Phe fluorescence postsynchronized to the high-low FRET transition at each codon (27 ribosomes).

Figure 4. Erythromycin stalls single translating ribosomes by blocking the nascent chain

Histograms of the number of complete FRET cycles for single ribosomes elongating on the 12F (left panels) and 6FK (right panels) mRNAs in the absence (blue bars) and presence (red bars) of erythromycin. Datasets are composed of 1034, 477, 1145, and 779 ribosomes for 12F, 12F plus erythromycin, 6FK, and 6FK plus erythromycin, respectively. Histograms are normalized to the number of ribosomes showing one FRET cycle.

Figure 5. The precise mechanisms and cumulative effects of ribosome-targeting antibiotics observed at codon resolution

(a) Comparison of mean lifetime estimations for all low-FRET states, the first high-FRET state, and subsequent high-FRET states in the absence (blue bars) or presence (red bars) of fusidic acid (301 molecules, top panel), and for all low-FRET and high-FRET states in the absence (blue bars) and presence (red bars) or either spectinomycin (middle panel) or viomycin (bottom panel) (333 and 351 molecules, respectively). All lifetimes are for productive events, defined as events followed by the next FRET state (e.g. high-FRET followed by low-FRET) (b) The relative efficiency of translation at each codon in the presence of fusidic acid, spectinomycin, and viomycin, as compared to translation in the absence of antibiotics.

Figure 6. Monitoring the elongation cycle during multiple rounds of translation reveals increased translocation rates beyond initial codons

Mean lifetime estimates for the high-FRET (locked) and low-FRET (unlocked) states of the ribosome at the first ten codons of the 12F mRNA (1034 molecules). Error bars are 95% confidence intervals from single-exponential fits. The schematics above each panel represent the global conformational transition represented by each set of FRET state lifetimes.

Figure 7. A general model for ribosome dynamics and function

Interpretation of single-molecule data within the current locking/unlocking model of ribosome function. In the locked state, tRNA are held in the classical configuration, and the L1 stalk is kept open. The stability of this conformation may preserve reading frame and allow precise manipulation of tRNA during selection and catalysis. Ribosome unlocking upon peptide bond formation permits classical↔hybrid and open↔closed fluctuations of the tRNA and L1 stalk, respectively, and promotes spontaneous ratcheting. Unlocking may also loosen the ribosome:mRNA interaction. These motions might facilitate movement of tRNA and mRNA during translocation or initiation. The action of EF-G returns the ribosome to the locked state. L1 opening during this transition may facilitate dissociation of the E-site tRNA.