Ensemble and single-molecule FRET studies of protein synthesis

Abstract

Protein synthesis is a complex, multi-step process that involves large conformational changes of the ribosome and protein factors of translation. Over the last decade, Förster resonance energy transfer (FRET) has become instrumental for studying structural rearrangements of the translational apparatus. Here, we discuss the design of ensemble and single-molecule (sm) FRET assays of translation. We describe a number of experimental strategies that can be used to introduce fluorophores into the ribosome, tRNA, mRNA and protein factors of translation. Alternative approaches to tethering of translation components to the microscope slide in smFRET experiments are also reviewed. Finally, we discuss possible challenges in the interpretation of FRET data and ways to address these challenges.

mRNAs and lncRNAs intrinsically form secondary structures with short end-to-end distances

“…The 5′ phosphate of the RNA was labeled using cystamine and a maleimide derivative of Cy5 (or Cy3, Click Chemistry) in the following steps as previously described 66 : (i) the 5′ γ-phosphate of RNA was reacted with 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, Thermo Scientific) and imidazole; (ii) the phosphorimidazolide derivative of RNA was reacted with cystamine; (iii) the product was reduced with TCEP to release the thiophosphate group, which was subsequently modified by Cy5 (or Cy3) maleimide for 2 h at room temperature (RT). Labeled RNA molecules were purified using a 1 mL G-25 spin column equilibrated with ddH 2 O.…”

Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins

“…Another limitation of these ensemble FRET measurements is that most time-dependent investigations occur out of equilibrium, where kinetic parameters must to be extracted from the system’s relatively gradual return to equilibrium [ 35 , 36 ]. Here, the molecular ensemble is synchronized using rapid equilibrium perturbation techniques (e.g., stopped-flow mixing), which imposes strict constraints on the temporal duration of the perturbation and/or the range of accessible time-scales.…”

Strategic labelling approaches for RNA single-molecule spectroscopy

“…Either a native or via site-directed mutagenesis inserted cysteine can be used for labelling. Although cysteine is relatively rare in the proteome, additional native cysteines in the protein sometimes need to be mutated to achieve site-specific labelling, preferably without perturbation of the proteins folding and function [82][83][84][85].…”