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* These authors contributed equally
Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.
Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide–ribosome–mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.
Translation in eukaryotic systems occurs predominantly through 7-methylguanosine (m7G) cap-dependent pathways1. Studies indicate that the initiation step of eukaryotic translation is rate-limiting and a common target for regulation2,3,4. Mechanisms of cap-dependent translation have been extensively studied using genetic5, biochemical6,7,8, structural9, and genomic10 bulk approac....
1. Generation of reporter mRNA
Following the protocol described enables the imaging of individual antibody interactions with nascent N-terminal-tagged polypeptides with single-molecule resolution during active cell-free translation of 3' end-tethered reporter mRNA (Figure 1). A minimal demonstration experiment is reported with the use of three synthetic mRNAs: LUC (encoding untagged luciferase), LUCFLAG (encoding 3xFLAG-tagged luciferase), and hp-LUCFLAG.......
In comparison to typical in vitro TIRF single-molecule experiments, single-molecule imaging with the assay described here is additionally complex due to the use of cell extract and a high concentration of fluorescently labeled antibody. Compared to the more common practice of one round of surface PEGylation, a second round of PEGylation (step 2) greatly reduces nonspecific antibody binding to detection surface15. The high concentration of diffusing fluorescent antibodies causes an extreme.......
This work was supported by the National Institutes of Health [R01GM121847]; the Memorial Sloan Kettering Cancer Center (MSKCC) Support Grant/Core Grant (P30 CA008748); and MSKCC Functional Genomics Initiative.
....Name | Company | Catalog Number | Comments |
100X oil objective, N.A. 1.49 | Olympus | UAPON 100XOTIRF | |
Acryamide/bis (40%, 19:1) | Bio-Rad | 161-0144 | |
Alkaline liquid detergent | Decon | 5332 | |
Aminosilane (N-(2-Aminoethyl)-3-Aminopropyltrimethoxysilane) | UCT Specialties, LLC | A0700 | |
Andor ixon Ultra DU 897V EMCCD | Andor | DU-897U-CSO-#BV | |
Andor Solis software | Andor | For controlling the Andor EMCCD | |
Band-pass filter | Chroma | 532/640/25 | |
Band-pass filter | Chroma | NF03-405/488/532/635E-25 | |
Biotin-PEG-SVA | Laysan Bio Inc | Biotin-PEG-SVA | |
Coenzyme A free acid | Prolume | 309-250 | |
Coolterm software | For controlling the syringe pump | ||
Desktop computer | Dell | For controlling the microscope, camera, stage, and pump. | |
Dichroic mirror | Semrock | R405/488/532/635 | |
Direct-zol RNA microprep 50RNX | Fisher Scientific | NC1139450 | |
Dual-Luciferase Reporter Assay System | Promega | E1910 | |
Epoxy | Devcon | 14250 | |
Firefly luciferin D-Luciferin free acid | Prolume | 306-250 | |
Glacial acetic acid | Fisher Scientific | BP1185500 | |
Hydrogen perioxide | Sigma-Aldrich | 216763-500ML | |
Immersion oil | Olympus | Z-81226A | Low auto-fluorescence |
Luciferase Assay System | Promega | E1500 | |
MEGASCRIPT T7 Transcription Kit | Thermo fisher | AM1334 | |
Methanol | Fisher Scientific | MMX04751 | |
Microscope | Olympus | IX83 | |
Microscope slide | Thermo Scientific | 3048 | |
Monoclonal anti-FLAG M2-Cy3 | Sigma-Aldrich | A9594 | |
mPEG-SVA | Laysan Bio Inc | mPEG-SVA-5000 | |
MS(PEG)4 | Thermo Scientific | 22341 | |
NaCl (5M) | Thermo Scientific | AM9760G | |
No 1.5 microscope Cover glass | Fisherband | 12-544-C | |
Olympus Laser, 532nm 100mM | Olympus digital Laser system | CMR-LAS 532nm 100mW | |
Olympus TirfCtrl software | Olympus | For controlling the laser intensity and incident angle | |
Optical table | TMC vibration control | 63-563 | With vibration isolation |
Phenol chloroform isoamyl alcohol mix | Sigma-Aldrich | 77617-100ml | |
Pierce RNA 3' End Biotinylation Kit | Thermo Scientific | 20160 | |
Potassium hydroxide pellets | Sigma-Aldrich | P1767-500G | |
Prior motorized XY translation stage | Prior | PS3J100 | |
Prior PriorTest software | Prior | For controlling the Prior motorized stage | |
Recombinant RNasin RNase Inhibitor | Promega | N2515 | |
Stage top Incubator | In vivo scientific (world precision Instruments) | 98710-1 | With a custom built acrylic cage |
Staining jar | Fisher Scientific | 08-817 | |
Streptavidin | Thermo Scientific | 43-4301 | |
Sulfuric acid | Fisher Scientific | A300212 | |
SYBR green II | Fisher Scientific | S7564 | |
Syringe | Hamilton | 1725RN | |
Syringe pump | Harvard apparatus | 55-3333 | |
Tris (1M), pH = 7.0 | Thermo Scientific | AM9850G | |
Ultrasonic Bath | Branson | CPX1800H | |
Urea | Sigma-Aldrich | U5378-500G | |
Vaccinia Capping system | New England Biolabs | M2080S | |
Zymo-Spin IC Columns | Zymo Research | C1004 |
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