Name: dual dna rulers to study the mechanism of ribosome translocation with single-nucleotide resolution
Uploaded: 2019-07-08T13:00:00
Description: Watch this Scientific Journal Video about Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution at JoVE.com

This work is supported by the US National Institutes of Health (R01GM111452, Y.W., S.X.). Y. W. acknowledges support from the Welch Foundation (E-1721).

1. Preparation of the ribosome complexes
<ol>
	<li>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.</li>
	<li>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(...

<ol>
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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...

Biomolecular displacement is a fundamental parameter in studying the mechanism of the related biological functions. One particular example is the ribosome translocation1,2, 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 able to reveal whether the mRNA is smoothly threaded through the ribosome, or there are intermediate steps in which the two sides have different step sizes leading to a kinked or looped mRNA conformation inside the ribosome.
Several methods have been developed to address the first challenge of resolving different steps on the exit side of the ribosome (the 3&#39;&#160;end of the mRNA). The dual luciferase assay resolves the different reading frames by measuring the ratios of the resulting different proteins3,4. It is only applicable for the 3&#39;&#160;end of the mRNA and thus insufficient to provide a complete picture of translocation. Mass spectrometry can analyze the different peptide fragments as the consequence of the corresponding code rearrangements5. But it cannot pinpoint to how many nt the ribosome moves on the mRNA. The toe-printing assay is another common method that uses a reverse transcriptase primed at the 3&#39;-distal end to transcribe the mRNA toward the ribosome6. However, it is not applicable for the 5&#39;&#160;end of mRNA that is entering the ribosome. Other techniques, including single molecule approaches and fluorescence methods7, are difficult to achieve single-nt resolution.
We have developed the dual DNA ruler assay that can uniquely determine both the entrance and exit positions of the uncovered mRNA in ribosome-mRNA complexes. &#160;The ruler DNAs are DNA oligomers that form duplexes of certain numbers of basepairs (bp) with the mRNA uncovered by the ribosome, regardless of which end of the mRNA. The bp numbers then precisely reveal the ribosome position on the mRNA during translocation. The bp numbers of the duplexes are determined by their critical dissociation forces obtained from force-induced remnant magnetization spectroscopy (FIRMS)8. With 2-3 pN force uncertainty, the critical forces are sufficient to offer single-nt resolution. By implementing a linker molecule on the DNA rulers, the sterically hindered side of the mRNA by the ribosome can be probed. Different ribosomal displacements can thus be accurately resolved. We have successfully revealed a unique looped conformation of mRNA trapped by antibiotics during translocation9, and resolved different reading frames that coexisted on a slippery mRNA sequence10. This article describes the details of the dual ruler assay, which include preparation of the ribosome complexes, surface functionalization of the glass slides, immobilization of the ribosome complexes and their hybridization with magnetically labeled DNA ruler molecules, magnetic detection, and force spectrum analysis by FIRMS.&#160;

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			<td height="21" style="height:21px;width:217px;">Styrene Strip</td>
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			<td style="width:239px;">Evaporated Coatings</td>
			<td style="width:161px;"></td>
			<td>60.0 &#215; 4.0 &#215; 0.3 mm3</td>
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			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">F0756-1G</td>
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			<td height="21" style="height:21px;width:217px;">Neomycin Sulfate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">1458009</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Viomycin Sulfate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">1715000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Hygromycin</td>
			<td style="width:239px;">invitrogen</td>
			<td style="width:161px;">10687-010</td>
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			<td height="21" style="height:21px;width:217px;">Tris-HCl</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">T5941-100G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Magnesium acetate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">M5661-50G</td>
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			<td height="21" style="height:21px;width:217px;">Ammonium chloride</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">A9434-500G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Potassium chloride</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P9333-500G</td>
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			<td height="21" style="height:21px;width:217px;">EDTA</td>
			<td style="width:239px;">GIBCO</td>
			<td style="width:161px;">774750</td>
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			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">M6250-500ML</td>
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			<td height="21" style="height:21px;width:217px;">Tween20</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P1379-250ML</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">GTP</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">G8877-100MG</td>
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			<td height="21" style="height:21px;width:217px;">PEP</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P7127-100MG</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">Pyruvate Kinase</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P1506-5KU</td>
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			<td height="21" style="height:21px;width:217px;">Sucrose&#160;</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">S7903-5KG</td>
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			<td height="21" style="height:21px;width:217px;">Dynabeads M-280 Streptavidin</td>
			<td style="width:239px;">ThermoFisher</td>
			<td style="width:161px;">11205D</td>
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			<td height="21" style="height:21px;width:217px;">mRNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">133899727</td>
			<td>5&#8242;-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&#8242;&#160;</td>
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			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">157468630</td>
			<td>3&#697;- TAA TTT AAT TTA ATT TTT CGA AAU AT50/TEGBio/-5&#697;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">164845370</td>
			<td>3&#697;-AAT TTA ATT TTT CCT TTA AAA AT50/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">157468628</td>
			<td>3&#697;-AAA ATC CCG CGT TAG AAC UGG GG/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">163472705</td>
			<td>3&#697;-CCG CGT TAG ATG ACG AGA ACG GG/TEGBio/-5&#8217;&#160;</td>
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			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678130</td>
			<td>3&#697;-AGA TGA CGA CTT CTC GGG/TEGBio/-5&#8217;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678131</td>
			<td>3&#697;-T AGA TGA CGA CTT CTC GGG/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678132</td>
			<td>3&#697;-TT AGA TGA CGA CTT CTC GGG/TEGBio/-5&#697;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678133</td>
			<td>3&#697;-GTT AGA TGA CGA CTT CTC GGG/TEGBio/-5&#8217;&#160;</td>
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			<td height="21" style="height:21px;width:217px;">Centrifuge</td>
			<td style="width:239px;">Eppendorf</td>
			<td style="width:161px;">5427R</td>
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			<td style="width:239px;">Hitachi</td>
			<td style="width:161px;">CS150FNX</td>
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			<td height="21" style="height:21px;width:217px;">Vortex mixer</td>
			<td style="width:239px;">VWR</td>
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			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">SR530</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Lock-in Amplifier</td>
			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">SR830</td>
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			<td style="width:161px;">TLB-6918-D</td>
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			<td style="width:239px;">Stanford Research Systems</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Photo detectors</td>
			<td style="width:239px;">Thorlabs</td>
			<td style="width:161px;">DET36A</td>
			<td></td>
		</tr>
	</tbody>
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dual dna rulers to study the mechanism of ribosome translocation with single-nucleotide resolution

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).&#160; 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.

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...

Watch this Scientific Journal Video about Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution at JoVE.com

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

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.

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

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