Synchronized translation for detection of temporal stalling of ribosome during single-turnover translation.
Arrhythmic translation caused by temporal stalling of ribosome during translation elongation is essential for gene expression and protein folding. To analyze the positions of the temporarily stalled ribosome and length of the stalling, the ribosomes must be synchronized during translation elongation. In this study, we designed a two-step translation reaction to synchronize the ribosome during a single-turnover translation. First, ribosomes decoding mRNA were artificially and specifically halted before isoleucine codon by reducing isoleucyl-tRNA synthetase from reaction mixture of in vitro translation. Then, translation elongation was restarted simultaneously to synchronize the translation. It enabled evaluation of translation elongation with time resolving capacity shorter than ever before. In addition, position-specific incorporation of fluorescent amino acid and mass spectrometry analyses enabled trace of translation elongation after gel electrophoresis and accurate determination of ribosome positions temporarily stalled before rare codons, respectively. The synchronized translation demonstrated here would be useful to evaluate trans- and cis-elements that affect rate of the translation elongation.
Tamaki
T
Endoh
Yu
Y
Kawasaki
Naoki
N
Sugimoto
D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D005786:Gene Expression Regulation; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
D003062:Codon; D012333:RNA, Messenger; D012343:RNA, Transfer; D007533:Isoleucine-tRNA Ligase