Fig. 2: Engineering 23S-cp5S rRNA.

a–c Secondary structures of the three engineered 23S-cp5S rRNA constructs DH42 (a), DH39 (b) and CH84 (c). 23S rRNA is in blue and integrated cp5S rRNA is in red; the connector linking 5′ and 3′ end of wt 5S rRNA is in green. d Schematic representation of the structures of wt and engineered 23S-cp5S rRNA operons. In cp5S, native 5′ and 3′ 5S rRNA ends are linked by a 4-nt connector (green). The resulting cp5S rRNA is “opened” in loop D (constructs DH42 and DH39) or loop C (construct CH84) and inserted via short tethers (dotted lines) connecting the indicated positions of the 23S rRNA and 5S rRNA. e Sucrose gradient separation of the ribosomal material from E. coli POP2136 cells expressing a mixed population of wt and 23S-cp5S ribosomes (construct DH42). The fractions used for preparation of the RNA from the large subunits (50S), ribosomes (70S), and polysomes are indicated. f Primer extension analysis of the 50S, 70S and polysomal rRNA prepared from randomly picked clones transformed with pDH42, pDH39, or pCH84 plasmids. In the presence of ddCTP, the primer is extended by 4 nt on the wt (A2058) rRNA template or by 3 nt on the 23S-cp5S rRNA template that contains the A2058G mutation. The first two lanes were loaded with the control samples: the radiolabeled primer (first lane) and primer extension products obtained with wt 23S rRNA. Note the presence of the mutant rRNA-specific band in the polysome fractions of the DH42 and CH84 constructs. The uncropped gel can be found in the Source data file. The results shown in e and f are typical representatives of 2 independent experiments.