Figure 2.

Schematic drawings of ribosomal complexes translating through the mRNA, and representative fluorescence time traces and corresponding E_FRET states. (A) Schematic drawings of ribosomal complexes during two rounds of translational elongation cycles on the dnaX mRNA monitored by single-molecule FRET experiments. A post-translocation ribosomal complex (POST-V) was enzymatically formed in bulk and contained peptidyl-tRNA^Val on the P-site. The complex was immobilized on the surface via hybridization at the 5΄-end with a surface-immobilized DNA strand. A pre-translocation complex (PRE-VK₁*), which contains tRNA^Val in the P-site and peptidyl-(Cy3)tRNA^Lys in the A-site, was formed on the surface by delivering a ternary complex of EF-Tu(GTP)-Lys(Cy3)tRNA^Lys in the absence of EF-G. PRE-VK₁* and PRE-K₁*K₂ complexes are likely in conformational fluctuations between classical and hybrid states. (B) Representative time traces of fluorescence signals of the dnaX-ribosomal complexes upon real-time (RT, black arrow) delivery of 100 nM (Cy5)EF-G to the PRE-VK₁*. The PRE-VK₁* complexes were identified by Cy3 signals at the beginning of imaging. Delivery of (Cy5)EF-G triggered excursions of high FRET states (red arrow), indicating accommodation of (Cy5)EF-G near the (Cy3)tRNA^Lys. Later on, Cy5 signals disappeared, while Cy3 intensity slightly increased. The observation is consistent with the published results that a slight increase of the Cy3 signal on the tRNA was assigned to the translocation of the tRNA from the A-site to the P-site (34). Slight increase of the Cy5 background signal is due to the floating (Cy5)EF-G. Bottom trace is a representative time trace of fluorescence signals of dnaX-ribosomal complexes upon co-delivery of (Cy5)EF-G and non-fluorescently labeled TC(K) to the PRE-VK₁*. First round of translocation (Tl1) by binding of (Cy5)EF-G1 (red arrow) and the second translocation (Tl2) process with (Cy5)EF-G2 bindings (blue arrows) and changes on the fluorescent signals are observed. Time traces were manually divided into two blocks for further analysis; block 1: from RT to Tl1, block 2: Tl1 to Tl2. (C) FRET histogram and transition density plot of block 1: from RT to Tl1. FRET state of ∼0.9 corresponds to EF-G bound state to PRE-VK₁*. (D) FRET histogram and transition density plot of block 2. FRET state of ∼0.3 corresponds to EF-G bound state to PRE-K₁*K₂. Transitions from 0 to 0.9 or 0.3 FRET correspond to binding of (Cy5)EF-G1 and (Cy5)EF-G2, while transitions from 0.9 or 0.3 to 0 correspond to dissociation of them, respectively.