Name: dual dna rulers to study the mechanism of ribosome translocation with single-nucleotide resolution
Uploaded: 2019-07-08T13:00:00.000+00:00
Duration: 10 min 27 s
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(enol)pyruvate.</li>
	<li>Incubate the five mixes separately at 37 &#176;C for 25 min before making the ribosome complexes. Prepare the five ribosome complexes as per Table 2. 
	NOTE: Post: post-translocation; Pre: pre-translocation.</li>
	<li>Add each of the five ribosome complexes onto a 1.1 M sucrose cushion separately, with volume ratio 1:1. Purify each with 450,000 &#215; g for 2 h in an ultra-centrifuge. Use a pipet to remove the supernatant and restore the ribosome complexes at -80 &#176;C after resuspension of the pellet with TAM10 buffer.</li>
</ol>
2. Preparation of biotin-coated glass slides
<ol>
	<li>Preliminary cleaning of the glass slides
	<ol>
		<li>Place 12 glass slides with dimensions of 60.0 &#215; 4.0 &#215; 0.3 mm3 (L &#215; W &#215; T) in a short and wide glass dish.</li>
		<li>Fill the glass dish with acetone and sonicate for 5 min. Then wash the slides with ultrapure water 5 times and fill &#190; of the dish with water.</li>
		<li>Add 10 M KOH to fill the dish and sonicate the glass slides for 20 min. Wash the slides with water 5 times.</li>
		<li>Add ethanol and sonicate for 5 min, pour out the ethanol and then dry them separately at 300 &#176;C for 3 h.</li>
	</ol>
	</li>
	<li>Aminosilane coating
	<ol>
		<li>Place the 12 cleaned slides back into the glass dish containing methanol. Clean a PEGylation flask with methanol by sonicating for 5 min, then fill it with 25 mL methanol, 1.25 mL water, 0.125 mL HAc, 0.25 mL 3-aminopropyltriethoxysilane (AMEO).</li>
		<li>Immediately replace the methanol in the glass dish with the prepared AMEO solution. Incubate at room temperature for 30 min.</li>
		<li>Rinse the slides with water several times, then dry them by nitrogen purge. Place the dried slides in clean glass dishes.</li>
	</ol>
	</li>
	<li>PEGlation
	<ol>
		<li>Prepare NaHCO3 solution (8.4 mg/mL) and PEGylation buffer (37.5 mg PEG, 6 mg biotinylated PEG, 150 &#956;L NaHCO3 solution). Mix them well by spinning at 6,000 rpm for 1 min.</li>
		<li>Place 25 &#956;L of PEG solution onto each slide. Cover it with the other slide on top. Make sure that there are no bubbles in between the two slides.&#160; Place the slides in an empty pipet tip box. Be sure that the box is leveled and place it in a dark drawer for about 3 h.</li>
		<li>Rinse the slides with water and dry again. Store the dried slides at room temperature under vacuum for up to 2 weeks.</li>
	</ol>
	</li>
</ol>
3. Sample preparation prior to magnetic and force measurements
<ol>
	<li>Machine a plastic sample well with dimensions 4 &#215; 3 &#215; 2 mm3 (L &#215; W &#215; D). Glue a piece of biotin-coated glass (approximately 5 mm long, cut from the 60 mm long slides prepared in section 2) on the bottom surface using epoxy.</li>
	<li>Add 20 &#956;L of 0.25 mg/mL streptavidin aqueous solution into the sample well and incubate at room temperature for 40 min. Then rinse the sample well twice with TAM10 buffer.</li>
	<li>Immobilize the ribosome complexes.
	<ol>
		<li>Without antibiotics: Use a pipette to remove buffer from the sample well, then add 20 &#956;L of 0.1 &#956;M ribosome complex (MF-Pre or MF-Post) into the sample well. The ribosome complex will bind with the streptavidin on the surface via the 5&#39;-end biotin on the mRNA. Incubate at 37 &#176;C for 1 h and then rinse once with TAM10 buffer.</li>
		<li>For the experiment using both neomycin and fusidic acid: incubate the MF-Pre complex with neomycin at 37 &#176;C for 10 min; incubate EF-G with fusidic acid at 37 &#176;C for 20 min. The concentrations are as follows: 0.1 &#181;M ribosome complex, 2 &#181;M EF-G, 4 mM GTP, 4 mM PEP, 0.02 mg/mL pyruvate kinase, 0.2 mM neomycin, and 0.25 mM fusidic acid.</li>
		<li>Carry out the other antibiotics experiments similarly. The concentrations are as follows: 0.2 mM viomycin, 0.4 mM hygromycin B, and 0.25 mM fusidic acid.</li>
		<li>For frameshifting study, repeat the above steps for the MFNF-Pre and MFNF-Post complexes involving the slippery motif U6A. Use antibiotics fusidic acid plus neomycin, and fusidic acid alone, respectively.</li>
	</ol>
	</li>
	<li>Remove buffer from the sample well, then add 20 &#956;L of 1 &#956;M biotinylated probing DNA strand and incubate at room temperature overnight. Rinse the formed DNA-mRNA duplex once with TAM10 buffer.</li>
	<li>Remove the buffer from the sample well. Then add 20 &#956;L of 0.5 mg/mL streptavidin-coated magnetic beads into the sample well and incubate at room temperature for 2 h.</li>
	<li>Carefully insert the sample well into a holder and place it in a centrifuge. Remove the free magnetic particles from the surface by centrifuging at 84 x g for 5 min.</li>
</ol>
4. Magnetic and force measurements
<ol>
	<li>Turning on the laser
	<ol>
		<li>Turn on the laser using the key. Then press the power button.</li>
		<li>Adjust the sensitivity of lock-in amplifier 1 (LIA1) to 500 mV and wait for about 2 h to warm up and stabilize the atomic magnetometer.</li>
	</ol>
	</li>
	<li>Setting up the atomic magnetometer
	<ol>
		<li>Run the instrument control software and set up the measurement parameters. Some parameters may slightly vary in each measurement. 
		NOTE: The atomic magnetometer comprises a laser (described above), an SR830 lock-in amplifier (referred to as LIA1), an SR530 lock-in amplifier (referred to as LIA2), DS345 (referred to as FG1) and ATF20B (referred to as FG2) function generators, a high-resolution motor and a computer. All of these should be turned on at this step of the protocol.</li>
		<li>Set up motor moving mode to Noise and default position to 0. Press Lock on front panel, adjust the sensitivity of LIA1 back to 200 mV.</li>
		<li>Adjust the current and voltage of the laser and find the proper resonance peak and signal-to-noise level. Press Sweep on the front panel. Note the amplitude/width ratio should be above 0.5 and phase value should be less than 5 degree. If not, re-do the sweep step.</li>
		<li>Plug-in the output of LIA2 to the feedback of the laser to lock its frequency. This amplifier measures the optical rotation of an auxiliary cesium cell to maintain the laser frequency on resonance12. The state remains until the end of the measurement.</li>
		<li>Plug-in function generator FG2 to input a square wave (500 mVpp, 100 mHz) as the reference signal. The square wave corresponds to 100 pT and is used to convert the current output of the amplifier to magnetic signal. Unplug the function generator.</li>
		<li>Set up motor moving mode to Two-way and default position to 260 mm. Check the status of the temperature controller to ensure the proper temperature (~37 &#176;C) of the atomic sensor.</li>
		<li>Check the stability of the whole system by measuring the signals of the empty sample holder twice. Evaluate stability and noise level after subtracting the two traces. Typical noise level and fluctuation should be &#177;2 pT at 30 ms integration time.</li>
	</ol>
	</li>
	<li>Magnetizing the sample
	<ol>
		<li>Gently place the sample on the magnetization station and let it stay for 2 min. 
		NOTE: The magnetization station consists of a permanent magnet (~0.5 T) and a plastic spacer.</li>
		<li>Put the sample back into the sample holder. Use 335.4 &#215; g centrifugal force to remove the nonspecifically bound magnetic particles.</li>
	</ol>
	</li>
	<li>Magnetic measurements after applying forces
	<ol>
		<li>Use tweezers to load the sample onto the motor. Click Lock on the front panel to run the program. Meanwhile, use tweezers to load the other sample in the holder and place it in the centrifuge. Note that the coated glass side should face the center of the centrifuge. 
		NOTE: The technique of force-induced remnant magnetization spectroscopy (FIRMS) is used, which uses an atomic magnetometer to measure the magnetic signal of the sample after applying mechanical force on the molecular interactions in the sample. Here, the molecular interactions are between the DNA ruler molecules and the mRNA in the ribosome complex. The force is increased stepwise by increasing the centrifugal speed. After applying each force, the motor translates the sample to the atomic sensor and then moves back. Hence, two magnetic field profiles are obtained, one during the forward scan and the other during backward scan13. We only use the latter to extract the peak height due to its better signal-to-noise ratio. The peak height in current (nA) is converted to magnetic signal amplitude (pT) based on the calibration square wave.</li>
		<li>Every measurement lasts approximately 5 min. When motor comes back to 0, click Save on the front panel. Carefully use tweezers to take samples from motor and centrifuge. Use initial speed corresponding to 335.4 &#215; g (2000 rpm, revolution per minute for the centrifuge listed in the Table of Materials).</li>
		<li>Exchange the two samples and apply a stronger force by increasing the centrifugal speed by 100 rpm or similar step size. Process alternately to gradually increase the force; a complete force spectrum is obtained after 10-12 data points.</li>
	</ol>
	</li>
	<li>Finishing the experiment
	<ol>
		<li>When all planned experiments are finished, turn off the equipment, proceeding in the opposite order as it was turned on.</li>
		<li>Remove the samples from the holder and immerse them in ethanol for cleaning and future use. Clean up the sample holder with acetone in case of magnetic beads contamination.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Open the Python analysis script and input all the experimental data. Click Load square to input the square wave as the reference. Then click Load baseline to input residual magnetic signal as background.</li>
		<li>Define the overall magnetic signal decrease as B0. Normalize each magnetic signal decrease B to B0 and express it as a percentage. Plot the percentage (B/B0) versus centrifugal force to obtain the FIRMS spectrum. 
		NOTE: The centrifugal force F is calculated from the buoyant mass of the magnetic beads m (4.6 &#215; 10&#8722;15 kg), centrifugal speed w, and radius of the centrifuge r (7.5 cm here) via equation F = mw2r. The typical force resolution is 2-4 pN and force range is 15-95 pN in this work.</li>
	</ol>
	</li>
</ol>

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No potential conflict of interest was reported by authors.

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;

<table><tbody><tr><td>Styrene Strip</td><td>City of Industry</td><td>MS-861</td><td></td></tr><tr><td>Glass slides</td><td>Evaporated Coatings</td><td></td><td>60.0 &amp;times; 4.0 &amp;times; 0.3 mm&lt;sup&gt;3&lt;/sup&gt;</td></tr><tr><td>Acetic acid</td><td>Millipore Sigma</td><td>A6283-500ML</td><td></td></tr><tr><td>3-Aminopropyltriethoxysilane</td><td>UCT specialties</td><td>21400088</td><td></td></tr><tr><td>mPEG-SVA</td><td>Laysan Bio</td><td>154-82</td><td></td></tr><tr><td>Biotin-PEG-SVA</td><td>Laysan Bio</td><td>152-84</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Millipore Sigma</td><td>S5761-500G</td><td></td></tr><tr><td>Epoxy glue</td><td>Devcon</td><td>31345</td><td></td></tr><tr><td>Streptavidin</td><td>ThermoFisher</td><td>434301</td><td></td></tr><tr><td>Fusidic Acid</td><td>Millipore Sigma</td><td>F0756-1G</td><td></td></tr><tr><td>Neomycin Sulfate</td><td>Millipore Sigma</td><td>1458009</td><td></td></tr><tr><td>Viomycin Sulfate</td><td>Millipore Sigma</td><td>1715000</td><td></td></tr><tr><td>Hygromycin</td><td>invitrogen</td><td>10687-010</td><td></td></tr><tr><td>Tris-HCl</td><td>Millipore Sigma</td><td>T5941-100G</td><td></td></tr><tr><td>Magnesium acetate</td><td>Millipore Sigma</td><td>M5661-50G</td><td></td></tr><tr><td>Ammonium chloride</td><td>Millipore Sigma</td><td>A9434-500G</td><td></td></tr><tr><td>Potassium chloride</td><td>Millipore Sigma</td><td>P9333-500G</td><td></td></tr><tr><td>EDTA</td><td>GIBCO</td><td>774750</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Millipore Sigma</td><td>M6250-500ML</td><td></td></tr><tr><td>Tween20</td><td>Millipore Sigma</td><td>P1379-250ML</td><td></td></tr><tr><td>GTP</td><td>Millipore Sigma</td><td>G8877-100MG</td><td></td></tr><tr><td>PEP</td><td>Millipore Sigma</td><td>P7127-100MG</td><td></td></tr><tr><td>Pyruvate Kinase</td><td>Millipore Sigma</td><td>P1506-5KU</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Millipore Sigma</td><td>S7903-5KG</td><td></td></tr><tr><td>Dynabeads M-280 Streptavidin</td><td>ThermoFisher</td><td>11205D</td><td></td></tr><tr><td>mRNA Oligo</td><td>Integrated DNA Technologies</td><td>133899727</td><td>5&amp;prime;-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&amp;prime;&amp;nbsp;</td></tr><tr><td>DNA Oligo</td><td>Integrated DNA Technologies</td><td>157468630</td><td>3?- TAA TTT AAT TTA ATT TTT CGA AAU AT50/TEGBio/-5?&amp;nbsp;</td></tr><tr><td>DNA Oligo</td><td>Integrated DNA Technologies</td><td>164845370</td><td>3?-AAT TTA ATT TTT CCT TTA AAA AT50/TEGBio/-5&amp;rsquo;&amp;nbsp;</td></tr><tr><td>DNA Oligo</td><td>Integrated DNA Technologies</td><td>157468628</td><td>3?-AAA ATC CCG CGT TAG AAC UGG GG/TEGBio/-5&amp;rsquo;&amp;nbsp;</td></tr><tr><td>DNA Oligo</td><td>Integrated DNA Technologies</td><td>163472705</td><td>3?-CCG CGT TAG ATG ACG AGA ACG GG/TEGBio/-5&amp;rsquo;&amp;nbsp;</td></tr><tr><td>DNA Oligo</td><td>Integrated DNA Technologies</td><td>138678130</td><td>3?-AGA TGA CGA CTT CTC GGG/TEGBio/-5&amp;rsquo;</td></tr><tr><td>DNA Oligo</td><td>Integrated DNA Technologies</td><td>138678131</td><td>3?-T AGA TGA CGA CTT CTC GGG/TEGBio/-5&amp;rsquo;&amp;nbsp;</td></tr><tr><td>DNA Oligo</td><td>Integrated DNA Technologies</td><td>138678132</td><td>3?-TT AGA TGA CGA CTT CTC GGG/TEGBio/-5?&amp;nbsp;</td></tr><tr><td>DNA Oligo</td><td>Integrated DNA Technologies</td><td>138678133</td><td>3?-GTT AGA TGA CGA CTT CTC GGG/TEGBio/-5&amp;rsquo;&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5427R</td><td></td></tr><tr><td>Micro Ultracentrifuge</td><td>Hitachi</td><td>CS150FNX</td><td></td></tr><tr><td>Vortex mixer</td><td>VWR</td><td>VM-3000</td><td></td></tr><tr><td>Lock-in Amplifier</td><td>Stanford Research Systems</td><td>SR530</td><td></td></tr><tr><td>Lock-in Amplifier</td><td>Stanford Research Systems</td><td>SR830</td><td></td></tr><tr><td>Laser</td><td>Newport</td><td>TLB-6918-D</td><td></td></tr><tr><td>Function generator</td><td>Stanford Research Systems</td><td>DS345</td><td></td></tr><tr><td>Photo detectors</td><td>Thorlabs</td><td>DET36A</td><td></td></tr></tbody></table>

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. ...

dual-dna-rulers-to-study-mechanism-ribosome-translocation-with-single

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

6.2K Views.  University of Houston. 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.

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

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Bioengineering

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences that encode rRNAs, composing regions called ribosomal DNA (rDNA) loci. Many organisms' genomes contain more than one rDNA locus distributed across different chromosomes, and rRNA may be transcribed from multiple rDNA loci or only a single locus. Changes in rRNA transcription sources often indicate ribosomal distress. However, the homogeneity of rRNA obstructs distinguishing if rRNA is transcribed from multiple or a single rDNA locus. Here, we describe a method that uses single nucleotide polymorphism-sensitive fluorescence in situ hybridization (SNP-FISH) to distinguish rRNA transcription between Drosophila melanogaster rDNA loci on the X and Y chromosomes. This method leverages locus-specific rRNA variants to enable easy detection of the source of rRNA transcription with single-cell resolution. This assay can be applied to any Drosophila cell type and may be adapted for use in other systems.

The protocol describes single nucleotide polymorphisms-sensitive fluorescence in situ hybridization (SNP-FISH) method to distinguish ribosomal RNA transcripts derived from the X or Y chromosome ribosomal DNA locus in Drosophila melanogaster.

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences ...

Genetics

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Biology

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100&#8722;200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100&#8722;200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...