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Figure 5 | BMC Molecular Biology

Figure 5

From: Requirements for resuming translation in chimeric transfer-messenger RNAs of Escherichia coli and Mycobacterium tuberculosis

Figure 5

In vitro tagging of a truncated ribosomal protein L27 by mutant M. tuberculosis tmRNAs in the presence of E. coli and M. tuberculosis SmpB proteins. (A) Chimeric tmRNAs M1, M2 and M3 were created by replacing three segments of M. tuberculosis tmRNA with corresponding segments of E. coli tmRNA. Four pseudoknots, the single-stranded portion of the open reading frame, the tRNA-like domain, helix 2 and helix 5 are denoted pk1-pk4, ssORF, TLD, hp2 and hp5, respectively. The single-stranded sequence connecting pk1 and ORF is marked as ‘up’. ORFs of E. coli and M. tuberculosis tmRNAs encode ANDE(H8) and ADSHQR(H6) peptide tags, respectively. (B) In vitro tagging of truncated ribosomal protein L27 by chimeric tmRNAs M1, M2 and M3 in the presence of either E. coli or M. tuberculosis SmpB proteins. Tagging was visualized by Western blotting with anti-T7 tag antibodies in ECL-Plex system. L27* denotes tagged ribosomal protein L27. (C) Graphical representation of Typhoon-derived data derived from four Western blotting analyses. Tagging efficiencies of hybrid tmRNA derivatives were normalized and compared to the tagging efficiency of the E. coli tmRNA(H8). Error bars show the standard deviation of three or more independent experiments.

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