Figure 11.

Real-time observation of translation using Zero-mode waveguides (ZMW).¹⁰¹ (A) Ribosomal initiation complex bearing fMet-(Cy3)tRNAfMet in the P-site was immobilized in a ZMW well, cylindrical aluminum nano-well (50–200 nm in diameter), enabling imaging in the presence of high concentrations of fluorophore-labeled molecules. (B) A representative trace of fluorescence intensities from a ribosomal complex translating six repeating Phe and Lys codons after initiation codon: M(FK)6. Ternary complexes (TC) with fluorescently labeled tRNAs, 200 nM Phe-(Cy5)tRNAPhe and 200 nM Lys- (Cy2)tRNALys, in the presence of 500 nM EF-G•GTP were delivered to the ribosomal initiation complex. Simultaneous illumination and detection of multi-color fluorophores Cy2 (blue), Cy3 (green), and Cy5 (red) allowed them to image tRNAs binding and release from the ribosomal complex during active translation elongation, thereby following translation codon by codon. Figure A and B are modified with permission from Figure 1 and 3A,