1. CCC.UGA: a new site of ribosomal frameshifting in Escherichia coli.
- Author
-
de Smit MH, van Duin J, van Knippenberg PH, and van Eijk HG
- Subjects
- Bacterial Proteins biosynthesis, Bacterial Proteins genetics, Base Sequence, Codon genetics, Gene Expression Regulation, Bacterial, Genes, Bacterial, Humans, Molecular Sequence Data, Mutagenesis, Insertional, Mutagenesis, Site-Directed, Plasmids, Protein Biosynthesis, Reading Frames, Recombinant Fusion Proteins biosynthesis, Transferrin genetics, Viral Proteins biosynthesis, Viral Proteins genetics, Escherichia coli genetics, Frameshift Mutation, Peptide Chain Termination, Translational, Ribosomes metabolism, Transferrin biosynthesis
- Abstract
To activate expression of a human transferrin (Tf)-encoding cDNA in Escherichia coli by translational coupling, it was placed in an expression 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 available mutants showed that this fusion results from +1 ribosomal frameshifting at the end of the R reading frame. This region contains the sequence, CCC.UGA, suggesting that before termination occurs, tRNA(Pro) may dislodge from the CCC codon and reassociate with the +1 triplet CCU. By further site-directed mutagenesis, we demonstrate that both the CCC codon and the termination codon are indeed required for the observed 2-4% frameshifting. When either triplet is changed, the frequency of frameshifting drops to 0.3% or less. These results classify CCC.UGA as a new '+1 shifty stop'.
- Published
- 1994
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