Structure
Volume 9, Issue 12, December 2001, Pages 1225-1236
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Article
Crystallographic and Modeling Studies of RNase III Suggest a Mechanism for Double-Stranded RNA Cleavage

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Abstract

Background: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg2+-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1–2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III.

Results: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn2+. The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg2+ ion, which provides the first glimpse of RNase III in action.

Conclusions: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a “ball-and-socket” junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn2+ binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

Keywords

RNase III
endonuclease domain
compound active center
dsRNA
RNA processing
RNA interference

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Correspondence: Xinhua Ji; e-mail: [email protected]