Programmed ribosomal frameshifting occurs at multiple sites and by multiple paths (109.3)

Programmed frameshifting is used by prokaryotes and eukaryotes to synthesize two or more proteins from the same messenger RNA. We have studied minus-one frameshifting in the dnaX gene in E. coli, whose mRNA contains the usual frameshifting signals: an internal Shine Dalgarno sequence, a slippery sequence (AAAAAAG), and a stem-loop. We used bulk mass spectrometry, single-molecule laser tweezers, and single-molecule FRET in our studies. We found that minus-one frameshifting occurs at the Lys codons in the slippery sequence, but also at codons on either side. Furthermore, the minus-one frameshift occurs by slips of the ribosome of -1, +2, or even -4 nucleotides. The translation trajectories show step-by-step progression as each codon is translated, but at the slippery sequence large-scale fluctuations in position of the ribosome are seen. This agrees with the mass spectrometry results showing multiple sites and multiple paths of frameshifting. After peptide bond formation the tRNAs undergo a classic-hybrid e...