CCC.UGA - A NEW SITE OF RIBOSOMAL FRAMESHIFTING IN ESCHERICHIA-COLI

To activate expression of a human transferrin(Tf)-encoding cDNA in Esc herichia coli by translational coupling, it was placed in an expressio n plasmid downstream from a 5'-terminal fragment from the replicase(R) -encoding gene of bacteriophage MS2. The resulting construct was found to produce, besides the desired Tf, a protein with the mobility of a fusion product (RTf) of the N-terminal R fragment and Tf Analysis of a vailable mutants showed that this fusion results from +1 ribosomal fra meshifting at the end of the R reading frame. This region contains the sequence, CCC.UGA, suggesting that before termination occurs, tRNA(Pr o) may dislodge from the CCC codon and reassociate with the +1 triplet CCU. By further site-directed mutagenesis, we demonstrate that both t he CCC codon and the termination codon are indeed required for the obs erved 2-4% frameshifting. When either triplet is changed, the frequenc y of frameshifting drops to 0.3% or less. These results classify CCC.U GA as a new '+1 shifty stop'.