∆ybeX cells accumulate rRNA fragments
As ybeX is located in the same operon with ybeY, whose role is implied in ribosome assembly, we assessed the rRNA profiles of WT Keio and ∆ybeZ , ∆ybeY , and ∆ybeX strains by formaldehyde denaturing agarose gel electrophoresis of total cellular RNA. In exponentially growing ∆ybeY cells, we saw a substantial accumulation of immature 16S rRNA (17S rRNA), while ∆ybeX and∆ybeZ cells had comparable levels of 17S rRNA to wild-type (Fig. 5A ). ∆ybeY cells also accumulate a faster-migrating 16S rRNA species, labelled as 16S* (Figure 5A;see also (Davies et al. , 2010)). When we assessed the RNA extracted from stationary phase cultures in ∆ybeX cells we observed a major RNA fragment of about a thousand nucleotides (Fig 5A ). This fragment was not present in material obtained from exponentially grown ∆ybeX cells. The wild type, the∆ybeZ and the ∆ybeY cells exhibited no such fragments in either stationary or exponential cells.
We used a more sensitive assay, the Northern blotting, on total RNA.Fig. 5B shows, for ∆ybeX lysates, a wide spectrum of 16S rRNA intermediates ranging from 500 nt (our lower detection limit) to almost full length 16S rRNA. Note that due to apparent cross-binding of our 16S-targeting probe to the 23S rRNA, we also see the 23S rRNAs as distinct bands in the gel, but importantly there are no degradation fragments between the full length 23S rRNA and 17S rRNA in any of the strains. Also, the 17S pre-rRNA is present for ∆ybeX and∆ybeY . Interestingly, ∆ybeX does not contain the 16S* rRNA species, which is present in ∆ybeY (but not ∆ybeX ). Except for the 16S* rRNA of ∆ybeY , the WT, ∆ybeZ and ∆ybeYlanes lack degradation intermediates. Thus, the ∆ybeX cells contain a unique and disparate mixture of 16S rRNA degradation intermediates.