PDBsum entry: 2eb1

Hydrolase

PDB id: 2eb1

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

Protein chains

Q9UPY3  (DICER_HUMAN) -  Endoribonuclease Dicer

Seq: 1922 a.a.

Struc: 171 a.a.

 Gene Ontology (GO) functional annotation 

• 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.