Analysis of translational termination of recombinant human methionyl-neurotrophin 3 in Escherichia coli.
A highly efficient UGA stop codon readthrough event during the synthesis of human neurotrophin 3 in E. coli is described. The incorporation of a Trp residue at the UGA stop codon is confirmed combining both the chemical analyses and the molecular and genetic data in this report. The 3' adjacent nuleotide to the UGA stop codon plays a crucial role in determining the readthrough efficiency in the order of A > G > C > U. The replacement of UGA with UAA or UAG totally abolished this readthrough phenomenon and the use of StpR host cells also prevented the occurrence of UGA readthrough. Gene dosage (or plasmid copy number) effect was not indicated in this event; however, the titration of RF-2 by mRNA transcripts under over-expression conditions might explain why tRNAtrp competes so well with RF-2 for UGA. Another apparently less produced readthrough product resulting from a transcript with no stop codon is also recorded, and the addition of a second in-frame stop codon increased the amount of the observed readthrough product.
Meng
S Y
SY
Department of Microbiology and Applied Microbial Genetics, Amgen Inc., Thousand Oaks, CA 91320-1789, USA.
Hui
J O
JO
Haniu
M
M
Tsai
L B
LB
D000595:Amino Acid Sequence; D001483:Base Sequence; D019208:Brain-Derived Neurotrophic Factor; D003062:Codon; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000302:isolation & purification; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D020933:Neurotrophin 3; D010443:Peptide Chain Termination, Translational; D010957:Plasmids; D017354:Point Mutation; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D019208:Brain-Derived Neurotrophic Factor; D003062:Codon; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020933:Neurotrophin 3; D012333:RNA, Messenger; D011994:Recombinant Proteins