Single-molecule imaging of full protein synthesis by immobilized ribosomes

Sotaro Uemura, Ryo Iizuka, Taro Ueno, Yoshihiro Shimizu, Hideki Taguchi, Takuya Ueda, Joseph D. Puglisi, Takashi Funatsu

Research output: Contribution to journalArticle

33 Citations (Scopus)

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.

Original languageEnglish
Article numbere70
JournalNucleic acids research
Volume36
Issue number12
DOIs
Publication statusPublished - 2008 Jul 1

ASJC Scopus subject areas

  • Genetics

Fingerprint Dive into the research topics of 'Single-molecule imaging of full protein synthesis by immobilized ribosomes'. Together they form a unique fingerprint.

  • Cite this

    Uemura, S., Iizuka, R., Ueno, T., Shimizu, Y., Taguchi, H., Ueda, T., Puglisi, J. D., & Funatsu, T. (2008). Single-molecule imaging of full protein synthesis by immobilized ribosomes. Nucleic acids research, 36(12), [e70]. https://doi.org/10.1093/nar/gkn338