The ΔybeX cells accumulate rRNA fragments
As ybeX is located in the same operon with ybeY, which is implicated in ribosome assembly, we assessed the rRNA profiles of total cellular RNA of the WT and the ΔybeZ , ΔybeY , andΔybeX strains by Northern blotting. For membrane hybridization, we used probes specific for the 17S precursor rRNA, the mature 16S rRNA and the 23S rRNA (Fig. 5A ).
In exponentially growing ΔybeY cells, we saw a substantial accumulation of immature 17S rRNA, while ΔybeX and ΔybeZcells had comparable levels of 17S rRNA to wild-type (Fig. 5B, D, E ). ΔybeY cells also accumulate a faster-migrating 16S rRNA species, labelled as 16S* (Fig. 5B; see also (Davies et al. , 2010)). The ΔybeX lysates do not contain the 16S* rRNA species.
When we assessed the total RNA extracted from stationary phaseΔybeX cell cultures, we observed an accumulation of 16S and 23S rRNA fragments (Fig. 5B, C ). These fragments were not present in material obtained from exponentially grown ΔybeX cells.Wild type, ΔybeZ and ΔybeY cells exhibited no such fragments in stationary or exponentially growing cells. BothΔybeX and ΔybeY stationary cells accumulate 17S rRNA (Fig. 5D, E ).
In addition, the stationary phase ΔybeX cells accumulate a wide spectrum of 17S pre-rRNA degradation intermediates ranging from a couple of hundred nucleotides to almost full-length 16S rRNA, as detected by the 17S 5‘-end specific oligonucleotide (Fig. 5D ). Fig. 5E , where we use a 3‘-end specific oligonucleotide, indicates that these decay intermediates lack the 17S 3‘-end. WT, ΔybeZ andΔybeY lysates lack such degradation intermediates. Thus, theΔybeX cells have a unique and disparate mixture of 17S rRNA and 16S rRNA degradation intermediates.