Ethidium-dependent uncoupling of substrate binding and cleavage by Escherichia coli ribonuclease III

Nucleic Acids Research
Irina Calin-JagemanAllen W Nicholson

Abstract

Ethidium bromide (EB) is known to inhibit cleavage of bacterial rRNA precursors by Escherichia coli ribonuclease III, a dsRNA-specific nuclease. The mechanism of EB inhibition of RNase III is not known nor is there information on EB-binding sites in RNase III substrates. We show here that EB is a reversible, apparently competitive inhibitor of RNase III cleavage of small model substrates in vitro. Inhibition is due to intercalation, since (i) the inhibitory concentrations of EB are similar to measured EB intercalation affinities; (ii) substrate cleavage is not affected by actinomycin D, an intercalating agent that does not bind dsRNA; (iii) the EB concentration dependence of inhibition is a function of substrate structure. In contrast, EB does not strongly inhibit the ability of RNase III to bind substrate. EB also does not block substrate binding by the C-terminal dsRNA-binding domain (dsRBD) of RNase III, indicating that EB perturbs substrate recognition by the N-terminal catalytic domain. Laser photocleavage experiments revealed two ethidium-binding sites in the substrate R1.1 RNA. One site is in the internal loop, adjacent to the scissile bond, while the second site is in the lower stem. Both sites consist of an A-A pair st...Continue Reading

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Citations

Oct 28, 2010·Journal of Plant Research·Eri KiyotaToshiyuki Fukuhara
Mar 29, 2003·Journal of Molecular Biology·Bruno LamontagneSherif Abou Elela
Jan 26, 2002·The International Journal of Biochemistry & Cell Biology·Christian Conrad, Reinhard Rauhut
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