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. 2018 Jun 5;46(16):8605–8615. doi: 10.1093/nar/gky488

Figure 5.

Figure 5.

Residue H122 of YqfG is critical for 16S 3′ processing activity and cell growth. (A) Spot dilution assay (log scale) of YqfG-depleted cells complemented with wt and various mutants (R59, H122 and R59/H122) of YqfG (strains CCB1051, CCB1033, CCB1034 and CCB1035, respectively). Mid-log phase cells were diluted to the same OD, then diluted in a 10-fold dilution series and 2 μl was spotted on plates with or without IPTG and containing either glucose or xylose. (B) Growth curves of the same strains in liquid 2xYT medium. Complementation with the wt strain is shown in blue, the R59A mutant in green, the H122A mutant in red and the R59A/H122A double mutant in orange. (C) Upper panel: total RNA isolated from the different complemented strains showing 16S and 23S rRNAs. Middle panel: northern blot of the different complemented strains probed with oligo CC172 showing accumulation of 16SS precursor species. Lower panel: northern blot of the different complemented strains probed with oligo CC172 showing release of the 65 nt processed fragment. (D) Sucrose gradients showing 30S, 50S and 70S peaks in the different complemented strains. The traces are colour coded as in panel B. (E) Western blot, probed with anti-His antibody, showing the levels of YqfG in the different complemented strains grown ±IPTG and in the presence of glucose or xylose. The migration positions of a molecular weight marker (in kDa) are shown to the left of the blot.