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PDBsum entry 2eb1

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protein metals Protein-protein interface(s) links
Hydrolase PDB id
2eb1
Jmol
Contents
Protein chains
171 a.a. *
Metals
_MG ×6
Waters ×192
* Residue conservation analysis
PDB id:
2eb1
Name: Hydrolase
Title: Crystal structure of thE C-terminal rnase iii domain of huma
Structure: Endoribonuclease dicer. Chain: a, b, c. Fragment: c-terminal rnase iii domain, rnase iii 2. Synonym: ribonuclease iii, helicase with rnase motif, helic engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.
Resolution:
2.00Å     R-factor:   0.212     R-free:   0.234
Authors: D.Takeshita,S.Zenno,W.C.Lee,K.Nagata,K.Saigo,M.Tanokura
Key ref:
D.Takeshita et al. (2007). Homodimeric structure and double-stranded RNA cleavage activity of the C-terminal RNase III domain of human dicer. J Mol Biol, 374, 106-120. PubMed id: 17920623 DOI: 10.1016/j.jmb.2007.08.069
Date:
05-Feb-07     Release date:   06-Nov-07    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
Q9UPY3  (DICER_HUMAN) -  Endoribonuclease Dicer
Seq:
Struc:
 
Seq:
Struc:
 
Seq:
Struc:
 
Seq:
Struc:
1922 a.a.
171 a.a.
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.3.1.26.3  - Ribonuclease Iii.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endonucleolytic cleavage to 5'-phosphomonoester.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     RNA processing   1 term 
  Biochemical function     RNA binding     2 terms  

 

 
DOI no: 10.1016/j.jmb.2007.08.069 J Mol Biol 374:106-120 (2007)
PubMed id: 17920623  
 
 
Homodimeric structure and double-stranded RNA cleavage activity of the C-terminal RNase III domain of human dicer.
D.Takeshita, S.Zenno, W.C.Lee, K.Nagata, K.Saigo, M.Tanokura.
 
  ABSTRACT  
 
Human Dicer contains two RNase III domains (RNase IIIa and RNase IIIb) that are responsible for the production of short interfering RNAs and microRNAs. These small RNAs induce gene silencing known as RNA interference. Here, we report the crystal structure of the C-terminal RNase III domain (RNase IIIb) of human Dicer at 2.0 A resolution. The structure revealed that the RNase IIIb domain can form a tightly associated homodimer, which is similar to the dimers of the bacterial RNase III domains and the two RNase III domains of Giardia Dicer. Biochemical analysis showed that the RNase IIIb homodimer can cleave double-stranded RNAs (dsRNAs), and generate short dsRNAs with 2 nt 3' overhang, which is characteristic of RNase III products. The RNase IIIb domain contained two magnesium ions per monomer around the active site. The distance between two Mg-1 ions is approximately 20.6 A, almost identical with those observed in bacterial RNase III enzymes and Giardia Dicer, while the locations of two Mg-2 ions were not conserved at all. We presume that Mg-1 ions act as catalysts for dsRNA cleavage, while Mg-2 ions are involved in RNA binding.
 
  Selected figure(s)  
 
Figure 4.
Figure 4. Activity of the RNase IIIb domain. (a) dsRNA cleavage activity of the RNase IIIb domain with 114 bp dsRNA substrates. The substrate was mixed with the RNase IIIb domain at 37 °C for 60 min, and resolved by electrophoresis in a 15% (w/v) polyacrylamide gel. Lane 1, dsRNA markers of 114 bp, 44 bp, and 21 bp; lane 2, no enzyme; lane 3, empty vector control; lanes 4–11, 0.1 ng μl^−1, 0.3 ng μl^−1, 1.0 ng μl^−1, 3.0 ng μl^−1, 10 ng μl^−1, 30 ng μl^−1, 100 ng μl^−1, and 300 ng μl^−1 (0.004 μM, 0.013 μM, 0.044 μM, 0.133 μM, 0.44 μM, 1.33 μM, 4.4 μM, and 13.3 μM, respectively) of RNase IIIb domain. (b) dsRNA cleavage activity of RNase IIIb-dsRBD with 114 bp dsRNA substrates. The substrate was mixed with RNase IIIb-dsRBD at 37 °C for 60 min, and resolved by electrophoresis in 15% (w/v) polyacrylamide gel. Lane 1, dsRNA markers of 114 bp, 44 bp, and 21 bp; lane 2, no enzyme; lane 3, empty vector control; lanes 4–11, 0.1 ng μl^−1, 0.3 ng μl^−1, 1.0 ng μl^−1, 3.0 ng μl^−1, 10 ng μl^−1, 30 ng μl^−1, 100 ng μl^−1, and 300 ng μl^−1 (0.003 μM, 0.01 μM, 0.032 μM, 0.095 μM, 0.32 μM, 0.95 μM, 3.2 μM, and 9.5 μM, respectively) of RNase IIIb-dsRBD. (c) dsRNA products of RNase IIIb, RNase IIIb-dsRBD and Ec-RNase III. The 114 bp dsRNA substrate was mixed with these enzymes at 37 °C for 60 min, and resolved by electrophoresis in a 20% (w/v) polyacrylamide gel. Lane 1, dsRNA markers of 114 bp, 44 bp, and 21 bp; lane 2, no enzyme; lane 3, empty vector control; lanes 4, 7, and 10, dsRNA markers of 21 bp, 15 bp, and 10 bp; lanes 5 and 6, 100 ng μl^−1 and 300 ng μl^−1 of RNase IIIb; lanes 8 and 9, 3.0 ng μl^−1 and 10 ng μl^−1 RNase IIIb-dsRBD; lane 11, 0.1 unit of Ec-RNase III (Ambion). (d) dsRNA cleavage activity of the RNase IIIb domain with the four different 21 bp dsRNA substrates (dsRNA substrates A, B, C, and D) ^32P-labeled at the 5′ end of either the upper (U*) or the lower (L*) strand. The upper images show the electrophoretic patterns using the substrates, which were incubated with RNase IIIb at 37 °C for 15 min. Lanes L and T1, alkaline-hydrolysis ladder and RNase T[1] products, respectively. Lanes S and –, substrate RNA and no enzyme control. Lane RIIIb is products of RNase IIIb, and lane oligo M is synthetic RNA oligonucleotide marker(s), and those sequences are shown on the right-hand side. RNase III generates products with 3′-OH termini, whereas alkaline hydrolysis and RNase T[1] generates fragments with 2′,3′-cyclic phosphate. Thus, synthetic RNA oligonucleotides with 3′-OH can be used for markers. The lower images show cleavage patterns of the substrates. Bold arrows indicate major cleavage sites and thin arrows indicate minor cleavage sites. These results indicate the RNase IIIb products with 2 nt 3′ overhang ends.
Figure 5.
Figure 5. Active sites of the RNase IIIb domain. (a) Stereoview of the electron density around the active site. 2F[o]–F[c] electron density maps contoured at 1.0 σ (blue) and 4.0 σ (orange) are shown around the magnesium ions and residues. (b) Active sites of the RNase IIIb domain, showing Mg-1, Mg-2, E1705, D1709, D1713, D1810, E1813, and water molecules. The hydrogen bonds and metal–ligand interactions are shown as broken lines. Left, the active sites of the homodimer between the molecules B and B', which are related by crystallographic symmetry. Right, the active sites of the homodimer between molecules A and C in the asymmetric unit. The conformation of the side-chain of D1713 in molecule C is different from those of molecules A and B, and is shown in blue. (c) Active sites of the RNase III domains (RNase IIIa and RNase IIIb) of Gi-Dicer, showing M1, M2, M3, E336, D340, V360, D404, E407, E649, D653, E673, E684, D720, and E723.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 374, 106-120) copyright 2007.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
22426548 P.W.Lau, K.Z.Guiley, N.De, C.S.Potter, B.Carragher, and I.J.MacRae (2012).
The molecular architecture of human Dicer.
  Nat Struct Mol Biol, 19, 436-440.  
21419681 E.S.Cenik, R.Fukunaga, G.Lu, R.Dutcher, Y.Wang, T.M.Tanaka Hall, and P.D.Zamore (2011).
Phosphate and R2D2 restrict the substrate specificity of Dicer-2, an ATP-driven ribonuclease.
  Mol Cell, 42, 172-184.  
19654583 C.F.Flores-Jasso, C.Arenas-Huertero, J.L.Reyes, C.Contreras-Cubas, A.Covarrubias, and L.Vaca (2009).
First step in pre-miRNAs processing by human Dicer.
  Acta Pharmacol Sin, 30, 1177-1185.  
19836333 P.W.Lau, C.S.Potter, B.Carragher, and I.J.MacRae (2009).
Structure of the human Dicer-TRBP complex by electron microscopy.
  Structure, 17, 1326-1332.  
19022417 V.Dincbas-Renqvist, G.Pépin, M.Rakonjac, I.Plante, D.L.Ouellet, A.Hermansson, I.Goulet, J.Doucet, B.Samuelsson, O.Rådmark, and P.Provost (2009).
Human Dicer C-terminus functions as a 5-lipoxygenase binding domain.
  Biochim Biophys Acta, 1789, 99.  
  19137112 E.Hefner, K.Clark, C.Whitman, M.A.Behlke, S.D.Rose, A.S.Peek, and T.Rubio (2008).
Increased potency and longevity of gene silencing using validated dicer substrates.
  J Biomol Tech, 19, 231-237.  
18927112 H.S.Soifer, M.Sano, K.Sakurai, P.Chomchan, P.Saetrom, M.A.Sherman, M.A.Collingwood, M.A.Behlke, and J.J.Rossi (2008).
A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs.
  Nucleic Acids Res, 36, 6511-6522.  
18268334 Z.Du, J.K.Lee, R.Tjhen, R.M.Stroud, and T.L.James (2008).
Structural and biochemical insights into the dicing mechanism of mouse Dicer: a conserved lysine is critical for dsRNA cleavage.
  Proc Natl Acad Sci U S A, 105, 2391-2396.
PDB codes: 3c4b 3c4t
18454937 d.o. .H.Lim, J.Kim, S.Kim, R.W.Carthew, and Y.S.Lee (2008).
Functional analysis of dicer-2 missense mutations in the siRNA pathway of Drosophila.
  Biochem Biophys Res Commun, 371, 525-530.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.