∆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.