Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Summary

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.

Abstract

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.

Introduction

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-tRNA^fMet 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 deacyl....

Protocol

1. Preparation of labeled ribosome and tRNA for FRET detection

1. Isolate ribosome without L27 from E. coli strain IW312 according to standard protocols^20,21. Extract the regular ribosome from E. coli strain MRE600.
2. Clone the L27'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 column.
3. Labeling of L27
   1. Incubate 20-100 μM of purified L27 in 100 μL with 2-10-fold excess of TCEP (Tris-2-carboxyethyl-p....

Results

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

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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