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.
Highlights
The final step in protein expression in cells is mRNAprogrammed synthesis of proteins by the ribosome
TRNAs, labeled with several different fluorophores, were detected binding to single ribosomes, using highly specialized instrumentation [10], suggesting that the sequence of the individual peptide being synthesized could be deduced from these signals
Fluorescence signals from labeled Phe-tRNAPhes, Val-tRNAVals and labeled ribosomal protein L1 were observed by total internal reflection fluorescence (TIRF) microscopy when the labeled tRNAs were bound to the surface immobilized ribosomes
Summary
The final step in protein expression in cells is mRNAprogrammed synthesis of proteins by the ribosome. Fluorescence methods for analyzing protein synthesis within intact cells are available, either via fusion of the target protein with fluorescent reporter proteins [3,4,5] or peptides that can be labeled with smaller, bright organic dyes [6,7,8,9]. These methods, though quite powerful, have significant limitations. TRNAs, labeled with several different fluorophores, were detected binding to single ribosomes, using highly specialized instrumentation [10], suggesting that the sequence of the individual peptide being synthesized could be deduced from these signals
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