(A) Position of the labeling sites in yeast modeled on the 3D structure of E. coli ribosome as viewed from inter-subunit interface. Small subunit is shown in wheat and large subunit is in teal. Surface-exposed loops replaced by hairpins are shown in red. H39 is located on the top of the head and H44 is proximal to the spur of the small subunit. H63 is at the bottom of the as viewed from inter-subunit interface. (B) rRNA from ribosomes bearing hairpin insertions was amplified by RT–PCR and sequenced. (C) Labeling hairpins and labeling oligonucleotides.

(A) Four nanomolar of H44 80S ribosomes were incubated with increasing concentrations of 5′-Cy3-sp68 oligonucleotide. Reactions were resolved on composite gel and apparent K_D was found to be 5 nm. (B) Labeled ribosomes were chased with 10-fold excess of dark sp68 oligonucleotide and incubated for up to 30 min at 30°C. No dissociation of the labeling nucleotide was detected. (C) The 1:1 labeling of the ribosomes with 5′-Cy3-sp68 oligonucleotide could be achieved by increasing incubation time to 15 min. Ten nanomolar ribosomes were incubated with increasing concentrations of 5′-Cy3-sp68 oligonucleotide. A 90–100% labeling efficiency could be reached at 1:1 ratio of the fluorescently labeled nucleotide to the ribosomes.

(A) Single-molecule fluorescence of biotin-mRNA:ribosome complexes immobilized on the surface of the quartz slide via biotin:neutravidin interactions. Ribosomes were labeled on the 40S subunit at H44 using Cy3-labeled nucleotide as described in the text. Fluorescence was excited by total internal reflectance illumination at 532 nm and detected using an EM-CCD camera. Preincubation of the slide with biotin abrogates complex binding. (B) Mean spot number in six fields of view.

(A) Single-molecule fluorescence trace from biotin-mRNA:Cy3-40S:Cy5-60S ribosomal complexes. (B) Lifetimes of the Cy3 and Cy5 dyes on the 40S and 60S ribosomal subunits.

Rupture trace. mRNA:80S ribosomal complexes are stretched between surface of the microscopic slide and polystyrene bead. Prior to measurement, the bead fluctuates around the attachment point. Bead capturing by an optical trap suppresses fluctuations. The piezoelectric stage is used to move surface of the slide, thus pulling the bead out of the focus of the trap. Bead displacement generates force linearly proportional to he displacement directed to the center of the optical trap. The force is increased until the complex ruptures.