ANALYSIS OF TRANSLATIONAL TERMINATION OF RECOMBINANT HUMAN METHIONYL-NEUROTROPHIN-3 IN ESCHERICHIA-COLI

A highly efficient UGA stop codon readthrough event during the synthes is 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 i n determining the readthrough efficiency in the order of A > G > C > U . The replacement of UGA with UAA or UAG totally abolished this readth rough phenomenon and the use of Stp(R) host cells also prevented the o ccurrence of UGA readthrough. Gene dosage (or plasmid copy number) eff ect was not indicated in this event; however, the titration of RF-2 by mRNA transcripts under over-expression conditions might explain why t RNA(trp) competes so well with RF-2 for UGA. Another apparently less p roduced readthrough product resulting from a transcript with no stop c odon is also recorded, and the addition of a second in-frame stop codo n increased the amount of the observed readthrough product. (C) 1995 A cademic Press. Inc.