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Real-time tRNA transit on single translating ribosomes at codon resolution.
- Source :
-
Nature [Nature] 2010 Apr 15; Vol. 464 (7291), pp. 1012-7. - Publication Year :
- 2010
-
Abstract
- Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we use zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically relevant micromolar ligand concentrations. Translation at each codon is monitored by stable binding of transfer RNAs (tRNAs)-labelled with distinct fluorophores-to translating ribosomes, which allows direct detection of the identity of tRNA molecules bound to the ribosome and therefore the underlying messenger RNA (mRNA) sequence. We observe the transit of tRNAs on single translating ribosomes and determine the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA molecule. Our results show that ribosomes are only briefly occupied by two tRNA molecules and that release of deacylated tRNA from the exit (E) site is uncoupled from binding of aminoacyl-tRNA site (A-site) tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.
- Subjects :
- Binding Sites
Escherichia coli
Fluorescence
Kinetics
Ligands
Luminescent Measurements
Optical Tweezers
Protein Biosynthesis genetics
RNA, Transfer genetics
Ribosomes chemistry
Ribosomes genetics
Time Factors
Codon genetics
Protein Biosynthesis physiology
RNA, Transfer metabolism
Ribosomes metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1476-4687
- Volume :
- 464
- Issue :
- 7291
- Database :
- MEDLINE
- Journal :
- Nature
- Publication Type :
- Academic Journal
- Accession number :
- 20393556
- Full Text :
- https://doi.org/10.1038/nature08925