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Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro
Author(s) -
Ramachandiran Vasanthi,
Kramer Gisela,
Horowitz Paul M,
Hardesty Boyd
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)02261-5
Subject(s) - ribosome , escherichia coli , transfer rna , shine dalgarno sequence , biology , codon usage bias , isoleucine , start codon , coding region , peptidyl transferase , biochemistry , synonymous substitution , genetics , translation (biology) , microbiology and biotechnology , stop codon , messenger rna , amino acid , gene , rna , leucine , genome
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.