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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 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' end of the mRNA). The dual luciferase assay resolves the different reading frames by measuring the ratios of the resulting different proteins^3,4. It is only applicable for the 3' 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'-distal end to transcribe the mRNA toward the ribosome⁶. However, it is not applicable for the 5' 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.  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. 

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Protokół

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 complexes as per Table 2.
   NOTE: Post: post-translocation; Pre: pre-translocation.
4. 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 × g for 2 h in an ultra-centrifuge. Use a pipet to remove the supernatant and restore the ribosome complexes at -80 °C after resuspension of the pellet with TAM10 buffer.

2. Preparation of biotin-coated glass slides

1. Preliminary cleaning of the glass slides
   1. Place 12 glass slides with dimensions of 60.0 × 4.0 × 0.3 mm³ (L × W × T) in a short and wide glass dish.
   2. Fill the glass dish with acetone and sonicate for 5 min. Then wash the slides with ultrapure water 5 times and fill ¾ of the dish with water.
   3. Add 10 M KOH to fill the dish and sonicate the glass slides for 20 min. Wash the slides with water 5 times.
   4. Add ethanol and sonicate for 5 min, pour out the ethanol and then dry them separately at 300 °C for 3 h.
2. Aminosilane coating
   1. 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).
   2. Immediately replace the methanol in the glass dish with the prepared AMEO solution. Incubate at room temperature for 30 min.
   3. Rinse the slides with water several times, then dry them by nitrogen purge. Place the dried slides in clean glass dishes.
3. PEGlation
   1. Prepare NaHCO₃ solution (8.4 mg/mL) and PEGylation buffer (37.5 mg PEG, 6 mg biotinylated PEG, 150 μL NaHCO₃ solution). Mix them well by spinning at 6,000 rpm for 1 min.
   2. Place 25 μL 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.  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.
   3. Rinse the slides with water and dry again. Store the dried slides at room temperature under vacuum for up to 2 weeks.

3. Sample preparation prior to magnetic and force measurements

1. Machine a plastic sample well with dimensions 4 × 3 × 2 mm³ (L × W × 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.
2. Add 20 μL 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.
3. Immobilize the ribosome complexes.
   1. Without antibiotics: Use a pipette to remove buffer from the sample well, then add 20 μL of 0.1 μM 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'-end biotin on the mRNA. Incubate at 37 °C for 1 h and then rinse once with TAM₁₀ buffer.
   2. For the experiment using both neomycin and fusidic acid: incubate the MF-Pre complex with neomycin at 37 °C for 10 min; incubate EF-G with fusidic acid at 37 °C for 20 min. The concentrations are as follows: 0.1 µM ribosome complex, 2 µM EF-G, 4 mM GTP, 4 mM PEP, 0.02 mg/mL pyruvate kinase, 0.2 mM neomycin, and 0.25 mM fusidic acid.
   3. 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.
   4. 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.
4. Remove buffer from the sample well, then add 20 μL of 1 μM biotinylated probing DNA strand and incubate at room temperature overnight. Rinse the formed DNA-mRNA duplex once with TAM₁₀ buffer.
5. Remove the buffer from the sample well. Then add 20 μL of 0.5 mg/mL streptavidin-coated magnetic beads into the sample well and incubate at room temperature for 2 h.
6. 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.

4. Magnetic and force measurements

1. Turning on the laser
   1. Turn on the laser using the key. Then press the power button.
   2. 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.
2. Setting up the atomic magnetometer
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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 °C) of the atomic sensor.
   7. 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 ±2 pT at 30 ms integration time.
3. Magnetizing the sample
   1. 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.
   2. Put the sample back into the sample holder. Use 335.4 × g centrifugal force to remove the nonspecifically bound magnetic particles.
4. Magnetic measurements after applying forces
   1. 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.
   2. 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 × g (2000 rpm, revolution per minute for the centrifuge listed in the Table of Materials).
   3. 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.
5. Finishing the experiment
   1. When all planned experiments are finished, turn off the equipment, proceeding in the opposite order as it was turned on.
   2. 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.
6. Data analysis
   1. 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.
   2. 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 × 10⁻¹⁵ 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.

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Wyniki

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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Dyskusje

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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Materiały

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