An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

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 regulation^2,3,4. Mechanisms of cap-dependent translation have been extensively studied using genetic⁵, biochemical^6,7,8, 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 pairs.
2. Synthesize untagged and tagged RNAs using linearized DNA templates and an in vitro transcription kit (see Table of Mater....

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), LUC_FLAG (encoding 3xFLAG-tagged luciferase), and hp-LUC_FLAG.......

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.......

Materials

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