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Nucleic Acids Res. 2008 Jul;36(12):e70. doi: 10.1093/nar/gkn338. Epub 2008 May 29.

Single-molecule imaging of full protein synthesis by immobilized ribosomes.

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1
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Abstract

How folding of proteins is coupled to their synthesis remains poorly understood. Here, we apply single-molecule fluorescence imaging to full protein synthesis in vitro. Ribosomes were specifically immobilized onto glass surfaces and synthesis of green fluorescent protein (GFP) was achieved using modified commercial Protein Synthesis using Recombinant Elements that lacked ribosomes but contained purified factors and enzyme that are required for translation in Escherichia coli. Translation was monitored using a GFP mutant (F64L/S65T/F99S/M153T/V163A) that has a high fluorophore maturation rate and that contained the Secretion Monitor arrest sequence to prevent dissociation from the ribosome. Immobilized ribosomal subunits were labeled with Cy3 and GFP synthesis was measured by colocalization of GFP fluorescence with the ribosome position. The rate of appearance of colocalized ribosome GFP was equivalent to the rates of fluorescence appearance coupled with translation measured in bulk, and the ribosome-polypeptide complexes were stable for hours. The methods presented here are applicable to single-molecule investigation of translational initiation, elongation and cotranslational folding.

PMID:
18511463
PMCID:
PMC2475623
DOI:
10.1093/nar/gkn338
[Indexed for MEDLINE]
Free PMC Article
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