Rewiring the ribosome to translate proteins encoded in its own RNA

a, RNA extracted from ribosomes isolated from E. coli BL21 cells transformed with pRibo-T, pRibo-TM(GFP), pRibo-TM(DFR) or Ribo-TM(HB) fractionated by agarose gel-electrophoresis and stained with ethidium bromide. The positions of the bands corresponding to 16S rRNA, 23S rRNA, and RNA originating from the Ribo-T or Ribo-TM ribosomes are indicated. M, DNA size markers. Note that mobility of double-stranded DNA markers does not match the mobility of single-stranded RNA. Shown is one of the two gels obtained in two independent experiments (n = 2 biological replicates) that produced similar results. b, The maps of the GFP-, DFR- and HiBiT ORFs appended to the 16S rRNA 3′ ends in various Ribo-TM construct and relative location of the primers used for RT-PCR; the expected sizes of the RT-PCR DNA products are indicated. c, Agarose electrophoresis of the RT-PCR products obtained with RNA isolated from the ribosomes prepared from cells transformed with the respective pRibo-TM plasmids (lanes 1–3), or the products of the PCR reaction generated with the same primers on the respective plasmid templates (lanes 4–6). The control PCR-only reactions carried out with the same RNA preparations as in lanes 1–3 were run in lanes 7–9. The RT-PCR bands with the correct sizes are marked with white dots; the authenticity of the RT-PCR products was verified by capillary sequencing. M, DNA size markers. Shown is one of the two gels obtained in two independent experiments (n = 2 biological replicates) that produced similar results. d, Growth of E. coli BL21 cells transformed with the original pRibo-TM(DFR) (quadrants marked ‘DFR’) or similar plasmids with the 16S or 23S rRNA mutations. Cells with pRibo-TM(DFR) containing mutations inactivating the small subunit (G530A in the 16S rRNA) or the large subunit (U2506A/U2585A double mutation in the 23S rRNA) are indicated as 16S* and 23S*, respectively. Cells transformed with Ribo-TM(GFP) (quadrants marked ‘GFP’) were used as a negative control. The plates contained either no Tmp (−Tmp), or 256 µg/ml of Tmp (+Tmp) and were incubated at 37 °C for the indicated time. e, MIC of Tmp for the GenR/EryR SQ110DTC6 cells transformed with pRibo-TM(DFR) or with pRibo-TM(GFP) as a control. Cells were grown in the absence or presence of Gen (256 µg/ml) and Ery (512 µg/ml). Three independent experiments yielded the same MIC values after 16 h incubation.