Fig. 3: The 23S-cp5S ribosomes support cell growth in the absence of free 5S rRNA.

a PCR analysis of rDNA in the E. coli SQA18 cells expressing wt 23S rRNA or chimeric 23S-cp5S rRNAs. Top: PCR with 23S rRNA-specific primers (black) flanking the site of cp5S rRNA (red) insertion into 23S rRNA (blue). Bottom: PCR with 5S rRNA-specific primers (black) annealing to the ends of wt 5S rRNA gene (red) generate no amplified product when DNA from the DH42 or CH84 clones is used as a template. b Polyacrylamide gel electrophoresis of total RNA or rRNA isolated from wt or SQA18/DH42 cells. Bottom: same samples as above but running electrophoresis for an extended time to improve separation of the large rRNA species. c Gel electrophoresis analysis of small RNA extracted from wt, SQA18/CH84, or SQA18/DH42 cells cured of the pCSacB plasmid. The gel was purposefully overloaded to confirm the lack of free 5S rRNA in the CH84 and DH42 clones. d Identification of the best linker combinations for the 23S-cp5S DH42 construct. The bar graph shows the fold enrichment of the indicated linker pairs in the selected library relative to the unselected one. e Growth of the E. coli SQA18 strain expressing wt ribosomes (black line) or ribosomes containing 23S-cp5S variants DH42(CUG/A) (referred as DH42*) (red line) and DH42(CG/0) (blue line). Each curve is an average of three replicates, with error bars indicating s.d. Calculated doubling time (τ) of the strains is indicated. The representative gels of at least two independent experiments are shown in a–c.