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Real-time tRNA transit on single translating ribosomes at codon resolution.

Uemura S, Aitken CE, Korlach J, Flusberg BA, Turner SW, Puglisi JD - Nature (2010)

Bottom Line: We observe the transit of tRNAs on single translating ribosomes and determine the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA molecule.Our results show that ribosomes are only briefly occupied by two tRNA molecules and that release of deacylated tRNA from the exit (E) site is uncoupled from binding of aminoacyl-tRNA site (A-site) tRNA and occurs rapidly after translocation.The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.

ABSTRACT
Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we use zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically relevant micromolar ligand concentrations. Translation at each codon is monitored by stable binding of transfer RNAs (tRNAs)-labelled with distinct fluorophores-to translating ribosomes, which allows direct detection of the identity of tRNA molecules bound to the ribosome and therefore the underlying messenger RNA (mRNA) sequence. We observe the transit of tRNAs on single translating ribosomes and determine the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA molecule. Our results show that ribosomes are only briefly occupied by two tRNA molecules and that release of deacylated tRNA from the exit (E) site is uncoupled from binding of aminoacyl-tRNA site (A-site) tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.

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Monitoring translation via fluorescent tRNA binding eventsa. Representative single-ZMW traces of ribosomes translating MFFF mRNA (top) and MFKF mRNA (bottom) in the presence of 30 nM EF-G and 30 nM TC. b. The number of fluorescent pulses observed in ZMWs depends on the presence of EF-G and TC. Event histograms for the three experiments in the absence (n=341) and presence (n=304) of 30 nM EF-G (top), and in the absence (n=278) and presence (n=297) of 30 nM unlabeled Lys-tRNALys TC (middle) and presence (n=355) of 30 nM Lys-(Cy2)-tRNALys TC (bottom). Histograms are normalized by the number of ribosomes showing single events.
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Figure 2: Monitoring translation via fluorescent tRNA binding eventsa. Representative single-ZMW traces of ribosomes translating MFFF mRNA (top) and MFKF mRNA (bottom) in the presence of 30 nM EF-G and 30 nM TC. b. The number of fluorescent pulses observed in ZMWs depends on the presence of EF-G and TC. Event histograms for the three experiments in the absence (n=341) and presence (n=304) of 30 nM EF-G (top), and in the absence (n=278) and presence (n=297) of 30 nM unlabeled Lys-tRNALys TC (middle) and presence (n=355) of 30 nM Lys-(Cy2)-tRNALys TC (bottom). Histograms are normalized by the number of ribosomes showing single events.

Mentions: Zero-mode waveguides (ZMWs, Fig. 1a) are nanophotonic confinement structures consisting of circular holes of 50-200nm diameter in a metal cladding film deposited on a solid, transparent substrate7. In conjunction with laser-excited fluorescence, ZMWs provide observation volumes on the order of zeptoliters (10-21 L), three to four orders of magnitude smaller than far-field excitation volumes. This drastically reduces the background signal from freely-diffusing fluorescent molecules, permitting the observation of fluorescent ligands in the μM range. Advances in fabrication8, surface chemistry9, and detection instrumentation10 have permitted direct monitoring of DNA polymerization in ZMWs11. The binding of labeled ligands to an enzyme immobilized in a ZMW is detected as a pulse of fluorescent light. Here we adapt this instrumentation to the study of translation. Using ZMWs, we observe real-time selection and transit of fluorescently-labeled tRNAs at μM concentration (Fig. 1b) on single ribosomes during multiple rounds of translation elongation. tRNA binding on single ribosomes was tracked using tRNAs that were specifically dye-labeled at their elbow positions without affecting their function12,13. Ribosomes were immobilized in ZMWs as 70S initiation complexes – containing fMet-(Cy3)tRNAfMet – assembled on biotinylated mRNAs, which were tethered to the biotin-PEG-derivatized bottom of ZMWs through neutravidin-biotin linkages; mRNAs contained 5’-UTR and Shine-Dalgarno sequences from T4 gene 32, an initiation codon and coding sequence of 3-12 codons, terminated by a stop (UAA) codon followed by four phenylalanine codons (Fig. 2a). Cy3 fluorescence from an immobilized complex confirmed the presence of initiator tRNA and marked a properly assembled and immobilized ribosome in a ZMW. The number of ribosome complexes immobilized per individual ZMW surfaces increased at higher ribosomal complex concentrations, obeying Poisson statistics, and, as expected, could be blocked by addition of free biotin (Fig. S1). Ellipsometry and ZMW experiments in the absence of ribosomes confirmed minimal nonspecific surface adsorption of translational components (100 μM tRNA, 1μM EF-Tu and EF-G)(Fig. S2).


Real-time tRNA transit on single translating ribosomes at codon resolution.

Uemura S, Aitken CE, Korlach J, Flusberg BA, Turner SW, Puglisi JD - Nature (2010)

Monitoring translation via fluorescent tRNA binding eventsa. Representative single-ZMW traces of ribosomes translating MFFF mRNA (top) and MFKF mRNA (bottom) in the presence of 30 nM EF-G and 30 nM TC. b. The number of fluorescent pulses observed in ZMWs depends on the presence of EF-G and TC. Event histograms for the three experiments in the absence (n=341) and presence (n=304) of 30 nM EF-G (top), and in the absence (n=278) and presence (n=297) of 30 nM unlabeled Lys-tRNALys TC (middle) and presence (n=355) of 30 nM Lys-(Cy2)-tRNALys TC (bottom). Histograms are normalized by the number of ribosomes showing single events.
© Copyright Policy - permission
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4466108&req=5

Figure 2: Monitoring translation via fluorescent tRNA binding eventsa. Representative single-ZMW traces of ribosomes translating MFFF mRNA (top) and MFKF mRNA (bottom) in the presence of 30 nM EF-G and 30 nM TC. b. The number of fluorescent pulses observed in ZMWs depends on the presence of EF-G and TC. Event histograms for the three experiments in the absence (n=341) and presence (n=304) of 30 nM EF-G (top), and in the absence (n=278) and presence (n=297) of 30 nM unlabeled Lys-tRNALys TC (middle) and presence (n=355) of 30 nM Lys-(Cy2)-tRNALys TC (bottom). Histograms are normalized by the number of ribosomes showing single events.
Mentions: Zero-mode waveguides (ZMWs, Fig. 1a) are nanophotonic confinement structures consisting of circular holes of 50-200nm diameter in a metal cladding film deposited on a solid, transparent substrate7. In conjunction with laser-excited fluorescence, ZMWs provide observation volumes on the order of zeptoliters (10-21 L), three to four orders of magnitude smaller than far-field excitation volumes. This drastically reduces the background signal from freely-diffusing fluorescent molecules, permitting the observation of fluorescent ligands in the μM range. Advances in fabrication8, surface chemistry9, and detection instrumentation10 have permitted direct monitoring of DNA polymerization in ZMWs11. The binding of labeled ligands to an enzyme immobilized in a ZMW is detected as a pulse of fluorescent light. Here we adapt this instrumentation to the study of translation. Using ZMWs, we observe real-time selection and transit of fluorescently-labeled tRNAs at μM concentration (Fig. 1b) on single ribosomes during multiple rounds of translation elongation. tRNA binding on single ribosomes was tracked using tRNAs that were specifically dye-labeled at their elbow positions without affecting their function12,13. Ribosomes were immobilized in ZMWs as 70S initiation complexes – containing fMet-(Cy3)tRNAfMet – assembled on biotinylated mRNAs, which were tethered to the biotin-PEG-derivatized bottom of ZMWs through neutravidin-biotin linkages; mRNAs contained 5’-UTR and Shine-Dalgarno sequences from T4 gene 32, an initiation codon and coding sequence of 3-12 codons, terminated by a stop (UAA) codon followed by four phenylalanine codons (Fig. 2a). Cy3 fluorescence from an immobilized complex confirmed the presence of initiator tRNA and marked a properly assembled and immobilized ribosome in a ZMW. The number of ribosome complexes immobilized per individual ZMW surfaces increased at higher ribosomal complex concentrations, obeying Poisson statistics, and, as expected, could be blocked by addition of free biotin (Fig. S1). Ellipsometry and ZMW experiments in the absence of ribosomes confirmed minimal nonspecific surface adsorption of translational components (100 μM tRNA, 1μM EF-Tu and EF-G)(Fig. S2).

Bottom Line: We observe the transit of tRNAs on single translating ribosomes and determine the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA molecule.Our results show that ribosomes are only briefly occupied by two tRNA molecules and that release of deacylated tRNA from the exit (E) site is uncoupled from binding of aminoacyl-tRNA site (A-site) tRNA and occurs rapidly after translocation.The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.

ABSTRACT
Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we use zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically relevant micromolar ligand concentrations. Translation at each codon is monitored by stable binding of transfer RNAs (tRNAs)-labelled with distinct fluorophores-to translating ribosomes, which allows direct detection of the identity of tRNA molecules bound to the ribosome and therefore the underlying messenger RNA (mRNA) sequence. We observe the transit of tRNAs on single translating ribosomes and determine the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA molecule. Our results show that ribosomes are only briefly occupied by two tRNA molecules and that release of deacylated tRNA from the exit (E) site is uncoupled from binding of aminoacyl-tRNA site (A-site) tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.

Show MeSH
Related in: MedlinePlus