Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

Summary

A protocol is presented for the production of tRNA(UUU) and the analysis of tRNA(UUU) in complex with the enzyme TcdA by agarose gel retardation assays.

Abstract

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N⁶-threonylcarbamoyladenosine (t⁶A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t⁶A (ct⁶A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 °C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Introduction

The gel retardation assay¹ (also known as electrophoretic mobility shift assay, EMSA) is an electrophoretic method designed to study and characterize protein-nucleic acid interactions. It allows the analysis of protein-DNA as well as protein-RNA interactions and in particular, interactions between tRNA and tRNA-binding proteins, using either purified components or complex mixtures of proteins (e.g., cell lysates) or nucleic acids (e.g., tRNA pools). We have applied gel retardation assays to the study of the interaction between purified tRNA(UUU) and TcdA, an N⁶-threonylcarbamoyladenosine (t⁶A) de....

Protocol

Caution: Consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in this protocol are toxic and corrosive. Use appropriate practices when performing the extraction of tRNA using phenol, including the use of a fume hood and personal protective equipment (safety glasses, gloves, lab coat, full-length pants, closed-toe shoes, etc.). This protocol uses a non-toxic nucleic acid stain. The use of alternative stains may require additional precautions and specialized disposal of the staining agent if toxic and/or carcinogenic (e.g., ethidium bromide).

1. Preparation of the Agarose Gel

Results

Large amounts of tRNA(UUU) can be obtained by expressing the tRNA gene in E. coli under the control of a strong inducible T7 promoter. The expressed tRNA(UUU) accumulates in the cytoplasm and is enriched over the pool of naturally abundant tRNAs. A two-step purification process consisting of a capture/extraction step and a gel filtration/polishing step was utilized to obtain EMSA-grade tRNA. The capture/extraction step uses pH 4.3 phenol to achieve the simultaneous precipitation .......

Discussion

The protein-tRNA agarose gel retardation assay described herein can be modified in a number of ways. First, the percentage of agarose in the gel can be reduced to allow the separation and visualization of protein-tRNA complexes significantly larger than the one analyzed here (120 kDa). Second, if the target protein is thermolabile, extra precautions must be taken to ensure that the temperature is kept below the maximum acceptable temperature by moving the electrophoresis apparatus to a cold room or by using ice packs ins.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
E. coli BL21(DE3)	Novagen	70235	DE3 lysogen contains T7 polymerase upon IPTG induction.
pET23d	Novagen	69748-3	pET23d confers resistance to ampicillin; T7 promoter.
LB Broth, Low Salt (Lennox L Broth), Granulated	Melford	GL1703	Conventional LB Broth, High Salt, can also be used.
Ampicillin	Sigma-Aldrich	10419313
Incubator Shaker (Thermostated)	NBS	Excella E24	Any shaker incubator with temperature control can be used.
Isopropyl-beta-D-thiogalactopyranoside (IPTG), >=99%	Acros Organics	BP1755
Sodium acetate, anhydrous, >99%	Melford	B4017
Ethylenediaminetetraacetic acid (EDTA), Disodium salt, >99%	Acros Organics	AC327205000	Harmful if inhaled.
Centrifuge (refrigerated)	Eppendorf	5430
Centrifuge (refrigerated) rotor	Eppendorf	5430/5430P
Centrifuge	Sorvall	RC5C
Centrifuge rotor	Sorvall	SLA-3000
Phenol solution, Saturated with 0.1 M citrate buffer, pH 4.3 ± 0.2	Sigma-Aldrich	P4682	Acute toxicity (oral, dermal, inhalation), corrosive
Ethanol absolute for analysis, 100% v/v	Merck Millipore	100983
Sodium phosphate dibasic dihydrate, Na2HPO4, >=99%	Sigma-Aldrich	71643
Potassium dihydrogen phosphate, KH2PO4	Merck	48,730,250
HiLoad 16/60 Superdex 75	GE Healthcare	28989333
Agarose	Melford	MB1200
Tris [Tris(hydroxymethyl) aminomethane HCl]	Melford	T1513
Boric Acid	Melford	B0503
Microwave	Haier	HDA-2070M
TCEP [Tris(2-carboxyethyl)phosphine hydrochloride]	Sigma-Aldrich	C4706	Corrosive to metals and skin.
ATP [Adenosine 5′-triphosphate disodium salt hydrate], Grade I, >=99%	Sigma-Aldrich	A2383
Magnesium chloride (MgCl2), anhydrous, >=98%	Sigma-Aldrich	M8266
Sodium chloride (NaCl), >99%	Fisher Bioreagents	BP358
Potassium chloride (KCl), >99%	Melford	P0515
NZYDNA Ladder VI	NZYtech	MB8901
Power supply	Consort	EV261
Electrophoresis unit	Real Laboratory	RELSMINI10
Real Safe nucleic acid staining	Real Laboratory	RBMSAFE	20,000X stock
UV Transilluminator (G:Box/EF)	Syngene	2216498
Coomassie Brilliant Blue G	Sigma-Aldrich	B0770
Rocker (Gyro-Rocker)	Stuart	SSL3
Mixer Vortex	Fisher brand	13214789