Protein tagging at rare codons is caused by tmRNA action at the 3′ end of nonstop mRNA generated in response to ribosome stalling

Effect of the number of consecutive rare codons on SsrA tagging. (A) Schematic drawing and sequences of the crp variants. The black box is the CRP ORF and the open box represents the region of rare codon inserted. The nucleotide sequence and amino acid sequence (in one-letter code) around this region are shown below the diagram. The AGG codons are underlined. (B) Western blot analysis of variant CRP proteins. Lysates equivalent to OD₆₀₀ = 0.01 prepared from TA341 (Δcrp), TA501 (Δcrp ΔssrA), or TA481 (Δcrp ssrA^DD) cells harboring the indicated plasmids were analyzed by Western blotting using anti-CRP and anti-DD-tag antibodies.