FRET-Based Identification of mRNAs Undergoing Translation

Abstract

We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

Dynamics of translation by single ribosomes through mRNA secondary structures

“…Fig. 2 also shows the fluorophore-labeled component(s) present in each complex and, qualitatively, the FRET efficiencies (blue halos) in the various complexes containing fluorophore pairs, which are in accord with previous results [18][19][20] .…”

“…We measured the dwell times of PRE and POST complexes that are formed as two elongation cycles add Arginine 8 and Phenylalanine 9 to the growing peptide, using three different smFRET measurements ( Fig. 2): i) between two adjacent tRNAs bound to the ribosome 18,19 ; ii) between tRNA and the large subunit protein, L11, near the A-site 18 ; and iii) between tRNA and L1, near the E-site 20 . The PRE and POST complexes studied in these experiments are shown in Fig.…”

“…Unlabeled 70S, 70S with Cy5-labeled L11 (70S-L11 Cy5 ), 50S with Cy5-labeled L1 (50S-L1 Cy5 ), and 30S ribosomes were prepared according to published procedures 18,20,37,38 . For tRNA-tRNA and L11-tRNA FRET experiments, initiation complexes were formed by mixture of 70S or 70S-L11 Cy5 ribosomes with mRNA, initiation factors, and fMet-tRNAf Met in TAM 15 buffer (37 °C, 25 min), and purified by centrifugation through a sucrose cushion 18 .…”

Elongation factor G initiates translocation through a power stroke

“…Ribosomes immobilized under these conditions successfully completed approximately six elongation cycles (Fig. S3), as detected by single-molecule fluorescence resonance energy transfer (FRET) patterns using Cy3-and Cy5-labeled tRNAs (25). Pol-TIRF recordings, including 16 different simultaneous polarized fluorescence intensities (PFIs) as shown in Fig.…”

Paving the way to single-molecule protein sequencing

“…The repurposed ClpXP showed a constant translocation speed and unidirectionality, features that are suitable for reliable fingerprinting. Note that a similar fingerprinting system was proposed and experimentally demonstrated by Goldman and colleagues, using a labelled ribosome to monitor the production of specific proteins inside the cell as a way to gain information on protein expression location and levels 37,38 . (Figure 2d).…”