Figure 4.

(a) Cleavage of total E. coli RNA by FLAG-Rne degradosomes. Total E. coli RNA was incubated in RNase E reaction buffer for 15 min at 30°C without (lane 1) or for 5 or 15 min with degradosomes (lanes 2 and 3). Samples for lanes 5–7 were prepared similarly, but reaction buffer was additionally supplemented with ATP and phosphate and incubation was done at 37°C. Lane 4 contains RNA components of degradosome. RNA was separated on gel and stained as described in Fig. 1a. Positions of 23S, 16S, and 5S rRNAs are shown. (b) Primer-extension analysis of degradosome RNase E cleavages. Total E. coli RNA was incubated in RNase E reaction buffer for 0 or 15 min without degradosomes (lanes 1 and 2) at 30°C. Incubations were performed for 15 min at the permissive temperature with degradosomes containing wild-type Rne (lane 3) or mutant (rne-3071) Rne (lane 4) or for 15 min at the nonpermissive temperature (44°C) with mutant Rne (lane 5). Lane 6 contains products from a primer-extension reaction of the RNA components isolated from FLAG-Rne degradosomes. Lanes 7–10 contain DNA sequencing ladder. Asterisks indicate fragments produced by RNase E. Aliquots of degradosome were incubated in RNase E buffer for 15 min at 30°C without addition of total RNA (lane 11). (c) Segments of folded 16S and 23S rRNAs (refs. 38 and 39) showing the identified cleavage sites.