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Nucleic Acids Research Advance Access originally published online on May 29, 2008
Nucleic Acids Research 2008 36(12):e70; doi:10.1093/nar/gkn338
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Nucleic Acids Research, 2008, Vol. 36, No. 12 e70
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Single-molecule imaging of full protein synthesis by immobilized ribosomes

Sotaro Uemura1,2, Ryo Iizuka1, Taro Ueno1, Yoshihiro Shimizu3, Hideki Taguchi3, Takuya Ueda3, Joseph D. Puglisi4 and Takashi Funatsu1,5,*

1Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, 2Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, 3Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa-shi, Chiba, 277-8562, Japan, 4Department of Structural Biology, Stanford University School of Medicine, USA 299 Campus Drive West, Stanford, CA 94305-5126, USA and 5Center for NanoBio Integration, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

*To whom correspondence should be addressed. Tel: +81-3-5841-4760; Fax: +81-3-5802-3339; Email: funatsu{at}mail.ecc.u-tokyo.ac.jp

Correspondence may also be addressed to Joseph D. Puglisi. Tel: 650-498-4397; Fax: 650-723-8464; Email: puglisi{at}stanford.edu

Received March 7, 2008. Revised May 8, 2008. Accepted May 9, 2008.

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.


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