title: The Functional Cycle of Rnt1p: Five Consecutive Steps of Double-Stranded RNA Processing by a Eukaryotic RNase III.
abstract: Double-stranded RNA (dsRNA)-specific RNase III proteins are required for RNA maturation and gene regulation. The mechanism of prokaryotic RNase IIIs has been well characterized, but how eukaryotic RNase IIIs (exemplified by Rnt1p, Drosha, and Dicer) work is less clear. Recently, we reported the crystal structure of Rnt1p in complex with RNA, revealing a double-ruler mechanism for substrate selection. Here, we present more structures of Rnt1p, either RNA free or RNA bound, featuring two major conformations of the enzyme. Using these structures with existing data, we describe the functional cycle of Rnt1p in five steps, selecting, loading, locking, cleavage, and releasing. We also describe atomic details of the two-Mg2+-ion catalytic mechanism that is applicable to all eukaryotic RNase III enzymes. Overall, our results indicate that substrate selection is achieved independent of cleavage, allowing the recognition of substrates with different structures while preserving the basic mechanism of cleavage.
authors.affiliation: Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: He
authors.initials: H
authors.lastname: Song
authors.affiliation: Structural Biophysics Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Xianyang
authors.initials: X
authors.lastname: Fang
authors.affiliation: Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Lan
authors.initials: L
authors.lastname: Jin
authors.affiliation: Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Gary X
authors.initials: GX
authors.lastname: Shaw
authors.affiliation: Structural Biophysics Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Yun-Xing
authors.initials: YX
authors.lastname: Wang
authors.affiliation: Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA. Electronic address: jix@mail.nih.gov.
authors.forename: Xinhua
authors.initials: X
authors.lastname: Ji
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D018360:Crystallography, X-Ray; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D002413:Cations, Divalent; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C408087:DICER1 protein, human; C517918:DROSHA protein, human; C468367:RNT1 protein, S cerevisiae; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases; D008274:Magnesium
keywords: RNA processing; RNase III; Rnt1p; SAXS; X-ray crystallography; double-stranded RNA; dsRNA; dsRNA processing; ribonuclease III; small-angle X-ray scattering