Name: single molecule fluorescence energy transfer study of ribosome protein synthesis
Uploaded: 2021-07-06T15:00:00
Description: Watch this Scientific Journal Video about Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis at JoVE.com

This work is supported by the US National Institutes of Health (R01GM111452) and the Welch Foundation (E-1721).

1. Preparation of labeled ribosome and tRNA for FRET detection
<ol>
	<li>Isolate ribosome without L27 from E. coli strain IW312 according to standard protocols20,21. Extract the regular ribosome from E. coli strain MRE600.</li>
	<li>Clone the L27&#39;s rpmA gene with C-terminal His-tag into pET-21b (+) plasmid, which is transformed and expressed in BL21(DE3)pLysS cells15. Purify the protein via a prepacked sepharose col...

<ol>
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SmFRET is sensitive to background signals. First, it is necessary to coat the sample chamber with 0.05% tween and then be added concurrently with the ribosome solution to block non-specific binding of the ribosome to the surface. To see fluorescence from the acceptor Cy5 emission, the oxygen scavenger cocktail (deoxy, glucose, and Trolox solutions) is essential. Without this solution, the bleaching is too fast in the acceptor channel to obtain useful information. Another critical step for ribosome experiments, specifical...

The ribosome is a &#248; 20 nm large ribonucleoprotein complex of a large (50S) and a small (30S) subunit. It assembles long peptides along the mRNA template processively and cooperatively. The ribosome 30S binds to the fMet-tRNAfMet and mRNA to start protein synthesis, and the 50S then joins to form the 70S initiation complex. The tRNAs bring amino acids to the ribosome at the A-site (aminoacyl- tRNA binding site), while the elongated peptidyl chain is held at the P-site (peptidyl- tRNA binding site). In the pre-translocation complex, the peptidyl chain is transferred to the tRNA at the A-site with one amino acid added. Meanwhile, the P-site tRNA is deacylated. Then, the A-, P- tRNAs move to the P-, E- sites to form the post-translocation complex, in which the E-site represents the tRNA exit site. In this state, the peptidyl-tRNA moves back to the P-site. The elongation cycle continues between the pre- and post-conformations while the ribosome translocates on the mRNA, one codon at a time1. The ribosome is highly coordinative of different functional sites to make this process efficient and accurate, such as inter-subunit ratcheting2, tRNA hybridization fluctuations3, GTPase activations4, L1 stalk opening-closing5, etc. Consequently, ribosomes quickly de-phase because every molecule moves at its own pace. The conventional methods can only deduce apparent average parameters, but low-populated or short-lived species will be masked in the average effect6. Single molecule method can break this limitation by detecting each ribosome individually, then identify different species via statistical reconstruction7. Different labeling sites have been implemented to probe ribosome dynamics, such as the interactions between tRNA-tRNA8, EF-G-L119, L1-tRNA10, etc. In addition, by labeling the large and small subunits, respectively, inter-subunit ratcheting kinetics and coordination with factors are observed11,12. Meanwhile, the smFRET method has broad applications in other central biological processes, and multi-color FRET methods are emerging13.
Previously a novel ribosome FRET pair was developed14,15. The recombinant ribosomal protein L27 has been expressed, purified, and labeled, and incorporated back into the ribosome. This protein interacted with the tRNAs at a close distance and helped stabilize the P-site tRNA in the post-translocation complex. When tRNA moved from the A- to the P-site, the distance between this protein and the tRNA is shortened, which can be distinguished by the smFRET signal. Multiple ribosome subpopulations have been identified using statistical methods and mutagenesis, and spontaneous exchange of these populations in the pre- but not post- translocation complex suggests the ribosome is more flexible before moving on the mRNA, and more rigid during decoding16,17,18. These variations are essential to the ribosome function. Here, the protocol describes the details of ribosome/tRNA-labeling, their incorporation in the ribosome, smFRET sample preparation, and data acquisition/analysis19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aminosilane</td>
			<td>Laysanbio</td>
			<td>MPEG-SIL-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEG</td>
			<td>Laysanbio</td>
			<td>Biotin-PEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS cells</td>
			<td>Novagen</td>
			<td>71403</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>millipore sigma</td>
			<td>C3515</td>
			<td></td>
		</tr>
		<tr>
			<td>CS150FNX Micro Ultracentrifuge</td>
			<td>nuaire</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3/C5-maleimide</td>
			<td>ApexBio</td>
			<td>A8138/A8140</td>
			<td></td>
		</tr>
		<tr>
			<td>ECLIPSE Ti2 inverted microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EdgeGARD Laminar Flow Hood</td>
			<td>Baker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>millipore sigma</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr>
			<td>Histrap HP column (Prepacked sepharose column)</td>
			<td>Cytiva</td>
			<td>17524701</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slip</td>
			<td>VWR</td>
			<td>48393-230</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope glass slides</td>
			<td>VWR</td>
			<td>470235-792</td>
			<td></td>
		</tr>
		<tr>
			<td>ORCA-Flash4.0 V3 camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PEG (5,000)</td>
			<td>Laysanbio</td>
			<td>MPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-21b (+) plasmid</td>
			<td>Novagen</td>
			<td>69741</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>VWR</td>
			<td>CPX-952-518R</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Apexbio</td>
			<td>B6055</td>
			<td></td>
		</tr>
		<tr>
			<td>Trolox</td>
			<td>millipore sigma</td>
			<td>238813</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The smFRET had the ribosome labeled at the middle position of tRNA traffic, to distinguish the tRNA translocation from the A- to the P-site (Figure 1)15. The distance from the L27 labeling residue to the A- or P-site tRNA is 52 or 61 &#197;, respectively, corresponding to FRET efficiency of 0.47 and 0.65. After the image collection, fluorescence intensities from the donor and acceptor channels were retrieved and plotted as time lapses (Figure 1</s...

Watch this Scientific Journal Video about Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis at JoVE.com

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

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 ...

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