CCC.UGA: a new site of ribosomal frameshifting in Escherichia coli.
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'.
de Smit
M H
MH
Department of Biochemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
van Duin
J
J
van Knippenberg
P H
PH
van Eijk
H G
HG
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D016368:Frameshift Mutation; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010443:Peptide Chain Termination, Translational; D010957:Plasmids; D014176:Protein Biosynthesis; D016364:Reading Frames; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012270:Ribosomes / Q000378:metabolism*; D014168:Transferrin / Q000096:biosynthesis* / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D003062:Codon; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D014764:Viral Proteins