Single-molecule imaging of full protein synthesis by immobilized ribosomes.
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.
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Sotaro
S
Uemura
Ryo
R
Iizuka
Taro
T
Ueno
Yoshihiro
Y
Shimizu
Hideki
H
Taguchi
Takuya
T
Ueda
Joseph D
JD
Puglisi
Takashi
T
Funatsu
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D014176:Protein Biosynthesis; D017510:Protein Folding; D012270:Ribosomes / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C497347:SecM protein, E coli; D014157:Transcription Factors; D049452:Green Fluorescent Proteins