Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

Summary

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

Abstract

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).  With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Introduction

Biomolecular displacement is a fundamental parameter in studying the mechanism of the related biological functions. One particular example is the ribosome translocation^1,2, during which the ribosome moves by exactly three nucleotides (nt) on the messenger RNA (mRNA) normally, and by one, two, or other numbers of nt except three in the case of frameshifting. Therefore, a molecular ruler system single-nt resolution is required to distinguish the different step sizes. A greater challenge is to probe the ribosome movement on both the entrance and exit sides. In other words, only with a dual ruler system will we be....

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Protocol

1. Preparation of the ribosome complexes

1. Make 1,000 mL of TAM10 buffer, which consists of 20 mM tris-HCl (pH 7.5), 10 mM Mg (OAc)₂, 30 mM NH₄Cl, 70 mM KCl, 5 mM EDTA, 7 mM BME (2-mercaptoethanol), and 0.05% Tween20.
2. Prepare the five mixtures listed in Table 1.
   NOTE: The ribosome was from the MRE600 strain¹¹. EF-Tu: elongation factor thermo unstable. EF-Ts: elongation factor thermo stable. GTP: guanosine triphosphate. PEP: phospho(enol)pyruvate.
3. Incubate the five mixes separately at 37 °C for 25 min before making the ribosome complexes. Prepare the five ribosome c....

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Results

Figure 1 shows the detection scheme and photographs of the major components. Magnetic detection is achieved by an atomic magnetometer using the scanning scheme (Figure 1A)¹³. The sample is placed on a rod mounted on a linear motor. The motor transports the sample to the atomic sensor inside a magnetic shield, then back to the original site for unloading. The atomic magnetometer detects the magnetic signal during the sampl.......

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Discussion

In our dual ruler assay, the magnetic beads play two essential roles. First, they serve as the force transducers because the centrifugal force is proportional to their buoyant mass. Second, the beads are signal carriers detected by an atomic magnetometer, which is currently the most accurate magnetic sensor. Combining mechanical manipulation and magnetic detection, the FIRMS technique is able to resolve a large number of molecular interactions based on their critical dissociation forces, which is the basis of the DNA rul.......

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Materials

Name	Company	Catalog Number	Comments
Styrene Strip	City of Industry	MS-861
Glass slides	Evaporated Coatings		60.0 × 4.0 × 0.3 mm3
Acetic acid	Millipore Sigma	A6283-500ML
3-Aminopropyltriethoxysilane	UCT specialties	21400088
mPEG-SVA	Laysan Bio	154-82
Biotin-PEG-SVA	Laysan Bio	152-84
Sodium bicarbonate	Millipore Sigma	S5761-500G
Epoxy glue	Devcon	31345
Streptavidin	ThermoFisher	434301
Fusidic Acid	Millipore Sigma	F0756-1G
Neomycin Sulfate	Millipore Sigma	1458009
Viomycin Sulfate	Millipore Sigma	1715000
Hygromycin	invitrogen	10687-010
Tris-HCl	Millipore Sigma	T5941-100G
Magnesium acetate	Millipore Sigma	M5661-50G
Ammonium chloride	Millipore Sigma	A9434-500G
Potassium chloride	Millipore Sigma	P9333-500G
EDTA	GIBCO	774750
2-mercaptoethanol	Millipore Sigma	M6250-500ML
Tween20	Millipore Sigma	P1379-250ML
GTP	Millipore Sigma	G8877-100MG
PEP	Millipore Sigma	P7127-100MG
Pyruvate Kinase	Millipore Sigma	P1506-5KU
Sucrose	Millipore Sigma	S7903-5KG
Dynabeads M-280 Streptavidin	ThermoFisher	11205D
mRNA Oligo	Integrated DNA Technologies	133899727	5′-Bio- CAA CUG UUA AUU AAA UUA AAU UAA AAA GGA AAU AAAA AUG UUU AAU UUU UUA GGG CGC AAU CUA CUG CUG AAC UC-3′
DNA Oligo	Integrated DNA Technologies	157468630	3?- TAA TTT AAT TTA ATT TTT CGA AAU AT50/TEGBio/-5?
DNA Oligo	Integrated DNA Technologies	164845370	3?-AAT TTA ATT TTT CCT TTA AAA AT50/TEGBio/-5’
DNA Oligo	Integrated DNA Technologies	157468628	3?-AAA ATC CCG CGT TAG AAC UGG GG/TEGBio/-5’
DNA Oligo	Integrated DNA Technologies	163472705	3?-CCG CGT TAG ATG ACG AGA ACG GG/TEGBio/-5’
DNA Oligo	Integrated DNA Technologies	138678130	3?-AGA TGA CGA CTT CTC GGG/TEGBio/-5’
DNA Oligo	Integrated DNA Technologies	138678131	3?-T AGA TGA CGA CTT CTC GGG/TEGBio/-5’
DNA Oligo	Integrated DNA Technologies	138678132	3?-TT AGA TGA CGA CTT CTC GGG/TEGBio/-5?
DNA Oligo	Integrated DNA Technologies	138678133	3?-GTT AGA TGA CGA CTT CTC GGG/TEGBio/-5’
Centrifuge	Eppendorf	5427R
Micro Ultracentrifuge	Hitachi	CS150FNX
Vortex mixer	VWR	VM-3000
Lock-in Amplifier	Stanford Research Systems	SR530
Lock-in Amplifier	Stanford Research Systems	SR830
Laser	Newport	TLB-6918-D
Function generator	Stanford Research Systems	DS345
Photo detectors	Thorlabs	DET36A