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 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 ribosome is a ø 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.
1. Preparation of labeled ribosome and tRNA for FRET detection
2. Preparation of the ribosome complexes
3. Preparation of sample slides
4. Single molecule FRET imaging
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 Å, 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). FRET efficiency was calculated by the formula Iacceptor/(Iacceptor+Idonor). A homemade program detected the single-step bleaching of the donor/acceptor and fitted the data before the bleaching points to avoid calculation of FRET from noises. After donor bleaching, both traces approached the baseline because no excitation can occur directly on the acceptor (Figure 2A). After acceptor bleaching, the donor intensity increased because fewer reaction pathways dissipate the excitation energy (Figures 2B,C).
It was found that the individual ribosomes exhibit different fluctuation, such as in the examples shown in Figure 2. These fluctuations are due to the wobbling motion of the tRNAs, which causes distance fluctuations to the L2717,18. In Figure 2, the dotted lines are the original data, and solid lines are the fitted data from the program. The fitted traces do not change the raw data reading but truncate the time-lapse traces after bleaching. The blue traces are the calculated FRET efficiencies. By tracking the exact same ribosomes after 5 min incubation at RT, it was found that one type of dynamics can switch to another23. Because these signals report on tRNA motions dictated by the surrounding ribosome, similar FRET efficiencies were grouped into various ribosome subpopulations.
Figure 3 shows very diverse subpopulations in the PRE complex. There are approximately 70% (640/951) of fluctuating species and 30% (311/951) of non-fluctuating species. These two categories can be further grouped into finer subpopulations. On the other hand, Figure 4 shows the opposite dynamics distribution. In POST, 65% of subpopulations are non-fluctuating, and the majority of them exhibited high FRET efficiency. These results indicate that the ribosome is more flexible in the PRE state than the POST state, which corroborates a cryo-EM study that concluded that the peptidyl chain at the P-site locks the ribosome dynamics in the POST state. However, in the PRE state, the peptidyl chain is transferred to the A-site, unlocking the ribosome and promoting5. The results supported the structure study conclusion under more physiological conditions. The unlocked state is correlated with the ratcheting motion between the 30S and 50S, and the locked state is correlated with the un-ratcheted conformation.
The subpopulation sorting method reveals the ribosome conformation upon inhibition by the antibiotic viomycin, as shown in Figure 514. The overall FRET efficiency histogram shows a major peak at 0.47, the classic state, without sorting. In this state, the ribosome is locked and un-ratcheted. However, sorting the subpopulations has revealed that 60% of the population is fluctuating as the unlocked PRE complex. Therefore, viomycin has trapped the ribosome at the ratcheted state. The results of this study contradicted an x-ray structure result at the time of publishing (2010) but were supported recently by another structure study (2020).
Figure 1: The labeling position of the Cy3/Cy5 on the ribosome and the tRNA. The distances between the labeling residue of L27 to the A- and P-site tRNA are shown (the red and blue stars show the approximate labeling locations of the Cy5 and Cy3, respectively). One representative time-lapse trace of fluorescence intensities from the CY3/Cy5 FRET pair is shown. A program detects bleaching points on donor/acceptor traces and truncates the trace before that point. This figure has been modified from Altuntop, M. E. et al.15. Please click here to view a larger version of this figure.
Figure 2: The typical ribosome traces were classified by their different dynamics. (A) A non-fluctuating ribosome trace. (B) A fluctuating ribosome trace that only samples FRET values lower than 0.6. (C) A fluctuating ribosome trace that samples FRET value higher than 0.6. The legends are shown in the plot. The fluorescence intensities of donor and acceptor are green and magenta, respectively. FRET values are calculated as Iacceptor/(Iacceptor+Idonor) and plotted separately in blue. The original data are displayed in dotted lines, and data truncated before the bleaching points are displayed in solid lines. This figure has been modified from Altuntop, M. E. et al.15. Please click here to view a larger version of this figure.
Figure 3: FRET efficiency histograms of the Pre-complex. The top-tier plots show the FRET histogram of the total ribosomes. The second-tier plots show the FRET histograms of the ribosomes separated into fluctuating (F) and non-fluctuating (NF) groups. The third-tier plots show the FRET histograms of the ribosomes further separated into groups of fluctuations that were above or below a FRET value of 0.6. Similar criteria were applied to the NF ribosomes to separate them into stable FRET states below or above 0.6 (NF-low, NF-high). The fourth-tier plots display the representative traces for each subpopulation. This figure has been modified from Altuntop, M. E. et al.15. Please click here to view a larger version of this figure.
Figure 4: FRET efficiency histograms of POST-Complex. The arrangement and grouping are similar to Figure 3. Contrary to Figure 3, the NF-High population is the majority. This figure has been modified from Altuntop, M. E. et al.15. Please click here to view a larger version of this figure.
Figure 5: Histograms of the PRE complex in the presence of 100 µM viomycin. The arrangement and grouping are similar to Figure 3. This figure has been modified from Ly, C. T. et al.14. Please click here to view a larger version of this figure.
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, specifically, is the PEG coating on the glass surface. The ribosome activity is sensitive to the surface environment; therefore, long brushing polymers are essential to shield unfavorable surface effects.
If the sample stage is too far from the focus, the auto-focusing system will not work. If this happens, turn off the auto-focus and manually adjust the objective position while observing the reflected laser spot. This is one advantage of a home-built total internal reflection illumination because the laser spot is visible. When the objective is near focus, the incident and reflected laser spots should be side by side, and the reflecting spot moves with the adjustment of the objective position. When these two spots are close, the auto-focus will work again.
One limitation of smFRET is the very low concentration range. Only up to 50 nM of fluorescently labeled substrates can be loaded without causing inhibiting background noise. Although surface-bound samples can require long-time acquisition on the same molecule, the time-resolution is limited to the ms range, while diffusion-based confocal smFRET can reach dynamics of the μs range24. Another limitation of the FRET method is the precise calculation of the distance from FRET efficiency. Due to different dye linkers and environments, the Forster distance varies from lab to lab. Therefore, comparing absolute distance can be problematic25. Although FRET efficiency change reveals the translocation mechanism, a more direct strategy was developed to measure the exact coverage of ribosome on the mRNA26,27.
Nevertheless, using FRET values as relative references to distinguish inhomogeneous populations within one experimental setting is a powerful method to reveal mechanism and dynamics one molecule at a time, which is not accessible with conventional methods. Furthermore, multi-color FRET and the combination of FRET with an optical trap will reveal more orchestrated ribosome dynamics in the future28. These developments are providing unprecedented sensitivity (displacement of Å distance) and new parameters (such as forces of pico-Newton magnitude) that are not achievable with existing methods28.
The authors have nothing to disclose.
This work is supported by the US National Institutes of Health (R01GM111452) and the Welch Foundation (E-1721).
Aminosilane | Laysanbio | MPEG-SIL-5000 | |
Biotin-PEG | Laysanbio | Biotin-PEG-SVA-5000 | |
BL21(DE3)pLysS cells | Novagen | 71403 | |
Catalase | millipore sigma | C3515 | |
CS150FNX Micro Ultracentrifuge | nuaire | ||
Cy3/C5-maleimide | ApexBio | A8138/A8140 | |
ECLIPSE Ti2 inverted microscope | Nikon | ||
EdgeGARD Laminar Flow Hood | Baker | ||
Glucose oxidase | millipore sigma | G2133 | |
Histrap HP column (Prepacked sepharose column) | Cytiva | 17524701 | |
Microscope cover slip | VWR | 48393-230 | |
Microscope glass slides | VWR | 470235-792 | |
ORCA-Flash4.0 V3 camera | Hamamatsu | ||
PEG (5,000) | Laysanbio | MPEG-SVA-5000 | |
pET-21b (+) plasmid | Novagen | 69741 | |
Sonicator | VWR | CPX-952-518R | |
TCEP | Apexbio | B6055 | |
Trolox | millipore sigma | 238813 |