FRET-based identification of mRNAs undergoing translation

PLoS One. 2012;7(5):e38344. doi: 10.1371/journal.pone.0038344. Epub 2012 May 31.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Feasibility Studies
  • Fluorescence Resonance Energy Transfer*
  • Fluorescent Dyes / metabolism
  • Molecular Sequence Data
  • Protein Biosynthesis*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism
  • Ribosomal Proteins / metabolism
  • Ribosomes / genetics
  • Ribosomes / metabolism

Substances

  • Fluorescent Dyes
  • RNA, Messenger
  • Ribosomal Proteins
  • ribosomal protein L1
  • RNA, Transfer