Ethidium-dependent uncoupling of substrate binding and cleavage by Escherichia coli ribonuclease III.
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 stacked on a CG pair, a motif which apparently provides a particularly favorable environment for intercalation. These results indicate an inhibitory mechanism in which EB site-specifically binds substrate, creating a cleavage-resistant complex that can compete with free substrate for RNase III. This study also shows that RNase III recognition and cleavage of substrate can be uncoupled and supports an enzymatic mechanism of dsRNA cleavage involving cooperative but not obligatorily linked actions of the dsRBD and the catalytic domain.
Calin-Jageman
I
I
Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Detroit, MI 48202, USA.
Amarasinghe
A K
AK
Nicholson
A W
AW
D001483:Base Sequence; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004722:Endoribonucleases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D004996:Ethidium / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D043244:Ribonuclease III
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D012330:RNA, Double-Stranded; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli; D004996:Ethidium