Figure 1
The AtYoeB toxin cleaves both RNA and DNA in vitro with the same concentration dependent efficiency. (a) AtYoeB possesses a sequence-independent RNase activity when incubated at 37 °C for 30 min; cleavage progresses in the presence of 2.5 mM MgCl2 (replicates given in Fig. S1), and while EDTA appears to slow the reaction, AtYoeB still degrades approximately 65% of the starting substrate indicating a metal-independent activity. (b) AtYoeB degrades DNA in a dose-dependent and sequence-independent manner, resulting in the accumulation of a nicked (N) topology under these reactions conditions. (SC, supercoiled; L, linear) (c) No DNA cleavage activity is present for the antitoxin AtYefM or for the complex of AtYoeB-YefM, demonstrating that DNA cleavage activity resides with the AtYoeB toxin. (d) Reactions performed using either DNA or RNA substrates in the presence of 2.5 mM MgCl2 and increasing AtYoeB were analyzed by electrophoretic methods (as in panels (a) and (b)). The intensity of either supercoiled DNA (n = 3) or total RNA (n = 3) were measured after electrophoresis and normalized relative to the control sample with no AtYoeB. (e) Quantitation of the dose dependence of RNA and DNA degradation in the presence of ribosomes (in vitro coupled transcription-translation) and absense of ribosomes reveals a much less efficient cleavage without the ribosome.
