FRET-based identification of mRNAs undergoing translation.
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.
Pennsylvania Muscle Institute, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Benjamin
B
Stevens
Chunlai
C
Chen
Ian
I
Farrell
Haibo
H
Zhang
Jaskiran
J
Kaur
Steven L
SL
Broitman
Zeev
Z
Smilansky
Barry S
BS
Cooperman
Yale E
YE
Goldman
D001483:Base Sequence; D005240:Feasibility Studies; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C028546:ribosomal protein L1; D012343:RNA, Transfer