Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro

Vasanthi Ramachandiran, Gisela Kramer, Paul M. Horowitz, Boyd Hardesty

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

The coding sequence for chloramphenicol acetyl transferase (CAT) contains several rare codons; three of them are ATA encoding isoleucine in positions 13, 84 and 119 of the amino acid sequence. Expression of CAT on Escherichia coli ribosomes in vitro results in mostly full-length product but also distinct smaller polypeptides from less than 3 kDa to over 20 kDa. As reported earlier, the smaller polypeptides are the predominant products, if translation is initiated with fluorophore-Met-tRNAf. All this translational pausing is eliminated when the first ATA codon is mutated to ATC, a frequently used codon for isoleucine in E. coli. Addition of large amounts of E. coli tRNA to the coupled transcription/translation reaction does not reduce the number of pause-site peptides seen in the expression of wild-type CAT. Thus we hypothesize that the mRNA structure may be an important determinant for translational pausing.

Original languageEnglish (US)
Pages (from-to)209-212
Number of pages4
JournalFEBS Letters
Volume512
Issue number1-3
DOIs
StatePublished - Feb 13 2002
Externally publishedYes

Keywords

  • In vitro protein synthesis
  • Nascent polypeptide
  • Rare codon
  • Translational pausing

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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