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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Generation of reporter mRNA

  1. Modify a DNA transcription template that encodes a bulk-assay “untagged” reporter mRNA by inserting an N-terminus epitope tag-encoding sequence to generate a DNA transcription template for a “tagged” reporter mRNA (Figure 2A).
    NOTE: 3xFLAG/anti-FLAG interaction is recommended for this assay due to its superior sensitivity and the short 3xFLAG tag length. However, the assay is compatible with other epitope/antibody pair.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
100X oil objective, N.A. 1.49OlympusUAPON 100XOTIRF
Acryamide/bis (40%, 19:1)Bio-Rad161-0144
Alkaline liquid detergentDecon5332
Aminosilane (N-(2-Aminoethyl)-3-Aminopropyltrimethoxysilane)UCT Specialties, LLCA0700
Andor ixon Ultra DU 897V EMCCDAndorDU-897U-CSO-#BV
Andor Solis softwareAndorFor controlling the Andor EMCCD
Band-pass filterChroma532/640/25
Band-pass filterChromaNF03-405/488/532/635E-25
Biotin-PEG-SVALaysan Bio IncBiotin-PEG-SVA
Coenzyme A free acidProlume309-250
Coolterm softwareFor controlling the syringe pump
Desktop computerDellFor controlling the microscope, camera, stage, and pump.
Dichroic mirrorSemrockR405/488/532/635
Direct-zol RNA microprep 50RNXFisher ScientificNC1139450
Dual-Luciferase Reporter Assay SystemPromegaE1910
EpoxyDevcon14250
Firefly luciferin D-Luciferin free acidProlume306-250
Glacial acetic acidFisher ScientificBP1185500
Hydrogen perioxideSigma-Aldrich216763-500ML
Immersion oilOlympusZ-81226ALow auto-fluorescence
Luciferase Assay SystemPromegaE1500
MEGASCRIPT T7 Transcription KitThermo fisherAM1334
MethanolFisher ScientificMMX04751
MicroscopeOlympusIX83
Microscope slideThermo Scientific3048
Monoclonal anti-FLAG M2-Cy3Sigma-AldrichA9594
mPEG-SVALaysan Bio IncmPEG-SVA-5000
MS(PEG)4Thermo Scientific22341
NaCl (5M)Thermo ScientificAM9760G
No 1.5 microscope Cover glassFisherband12-544-C
Olympus Laser, 532nm 100mMOlympus digital Laser systemCMR-LAS 532nm 100mW
Olympus TirfCtrl softwareOlympusFor controlling the laser intensity and incident angle
Optical tableTMC vibration control63-563With vibration isolation
Phenol chloroform isoamyl alcohol mixSigma-Aldrich77617-100ml
Pierce RNA 3' End Biotinylation KitThermo Scientific20160
Potassium hydroxide pelletsSigma-AldrichP1767-500G
Prior motorized XY translation stagePriorPS3J100
Prior PriorTest softwarePriorFor controlling the Prior motorized stage
Recombinant RNasin RNase InhibitorPromegaN2515
Stage top IncubatorIn vivo scientific (world precision Instruments)98710-1With a custom built acrylic cage
Staining jarFisher Scientific08-817
StreptavidinThermo Scientific43-4301
Sulfuric acidFisher ScientificA300212
SYBR green IIFisher ScientificS7564
SyringeHamilton1725RN
Syringe pumpHarvard apparatus55-3333
Tris (1M), pH = 7.0Thermo ScientificAM9850G
Ultrasonic BathBransonCPX1800H
UreaSigma-AldrichU5378-500G
Vaccinia Capping systemNew England BiolabsM2080S
Zymo-Spin IC ColumnsZymo ResearchC1004

References

  1. Hinnebusch, A. G. The scanning mechanism of eukaryotic translation initiation. Annual Review of Biochemistry. 83, 779-812 (2014).
  2. Gebauer, F., Hentze, M. W. Molecular mechanisms of translational control. Nature R....

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