title: Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro.
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-tRNA(f). 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas, Austin, TX, USA.
authors.forename: Vasanthi
authors.initials: V
authors.lastname: Ramachandiran
authors.forename: Gisela
authors.initials: G
authors.lastname: Kramer
authors.forename: Paul M
authors.initials: PM
authors.lastname: Horowitz
authors.forename: Boyd
authors.initials: B
authors.lastname: Hardesty
mesh_terms: D001483:Base Sequence; D002474:Cell-Free System; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics*; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007532:Isoleucine / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010441:Peptide Chain Elongation, Translational; D012270:Ribosomes / Q000378:metabolism
chemical_list: D003062:Codon; D007532:Isoleucine; D015500:Chloramphenicol O-Acetyltransferase