PDBsum entry 2g8h

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protein dna_rna ligands metals links
Hydrolase/RNA/DNA PDB id
Jmol PyMol
Protein chain
136 a.a. *
_MG ×2
Waters ×235
* Residue conservation analysis
PDB id:
Name: Hydrolase/RNA/DNA
Title: B. Halodurans rnase h catalytic domain d192n mutant in complex with mg2+ and RNA/DNA hybrid (non-p nick at the active site)
Structure: 5'-r( Up Cp Gp Ap Cp A)-3'. Chain: b. Engineered: yes. 5'-d( Ap Tp Gp Tp Cp G)-3'. Chain: c. Engineered: yes. Ribonuclease h. Chain: a. Fragment: bh-rnase hc.
Source: Synthetic: yes. Bacillus halodurans. Organism_taxid: 86665. Gene: rnha. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Trimer (from PQS)
1.85Å     R-factor:   0.206     R-free:   0.254
Authors: M.Nowotny,W.Yang
Key ref:
M.Nowotny and W.Yang (2006). Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release. EMBO J, 25, 1924-1933. PubMed id: 16601679 DOI: 10.1038/sj.emboj.7601076
02-Mar-06     Release date:   25-Apr-06    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
Q9KEI9  (RNH1_BACHD) -  Ribonuclease H
196 a.a.
136 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Biochemical function     nucleic acid binding     1 term  


DOI no: 10.1038/sj.emboj.7601076 EMBO J 25:1924-1933 (2006)
PubMed id: 16601679  
Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release.
M.Nowotny, W.Yang.
In two-metal catalysis, metal ion A has been proposed to activate the nucleophile and metal ion B to stabilize the transition state. We recently reported crystal structures of RNase H-RNA/DNA substrate complexes obtained at 1.5-2.2 Angstroms. We have now determined and report here structures of reaction intermediate and product complexes of RNase H at 1.65-1.85 Angstroms. The movement of the two metal ions suggests how they may facilitate RNA hydrolysis during the catalytic process. Firstly, metal ion A may assist nucleophilic attack by moving towards metal ion B and bringing the nucleophile close to the scissile phosphate. Secondly, metal ion B transforms from an irregular coordination in the substrate complex to a more regular geometry in the product complex. The exquisite sensitivity of Mg(2+) to the coordination environment likely destabilizes the enzyme-substrate complex and reduces the energy barrier to form product. Lastly, product release probably requires dissociation of metal ion A, which is inhibited by either high concentrations of divalent cations or mutation of an assisting protein residue.
  Selected figure(s)  
Figure 5.
Figure 5 Structure of the E188A Bh-RNase HC product complex. (A) Stereo view of the active site superimposed with the 2F[o]-F[c] map contoured at 0.9 . The 5'-phosphate is shown as red and yellow ball-and-sticks, Mg^2+ ions as purple spheres labeled with 'A' and 'B', and water molecules as small red spheres. (B, C) Side-by-side comparison of metal ion coordination in the substrate and product complexes. The substrate complex (D192N, PDB: 1ZBL) is shown in green with oxygen atoms in red and nitrogen atoms in blue, and the product complex (E188A) is shown in orange. (D) Stereo view of the superposition of substrate and product complexes.
Figure 6.
Figure 6 Schematic representation of the reaction steps proposed for RNase H. The substrate RNA is shown in pink and products in purple. Coordination of metal ions is highlighted in dark blue, and scissile phosphate in red. Selected hydrogen bonds are shown as blue lines. Black circles represent water molecules. The distance between the two metal ions is indicated in the enzyme–substrate, enzyme–intermediate and enzyme–product complexes.
  The above figures are reprinted by permission from Macmillan Publishers Ltd: EMBO J (2006, 25, 1924-1933) copyright 2006.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21109524 M.Ghosh-Kumar, T.I.Alam, B.Draper, J.D.Stack, and V.B.Rao (2011).
Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4.
  Nucleic Acids Res, 39, 2742-2755.  
20854710 W.Yang (2011).
Nucleases: diversity of structure, function and mechanism.
  Q Rev Biophys, 44, 1.  
20672157 B.Elsässer, and G.Fels (2010).
Atomistic details of the associative phosphodiester cleavage in human ribonuclease H.
  Phys Chem Chem Phys, 12, 11081-11088.  
20485342 B.H.Schmidt, A.B.Burgin, J.E.Deweese, N.Osheroff, and J.M.Berger (2010).
A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases.
  Nature, 465, 641-644.
PDB codes: 3l4j 3l4k
19782103 C.S.Adamson, and E.O.Freed (2010).
Novel approaches to inhibiting HIV-1 replication.
  Antiviral Res, 85, 119-141.  
20408915 E.Kanaya, T.Sakabe, N.T.Nguyen, S.Koikeda, Y.Koga, K.Takano, and S.Kanaya (2010).
Cloning of the RNase H genes from a metagenomic DNA library: identification of a new type 1 RNase H without a typical active-site motif.
  J Appl Microbiol, 109, 974-983.  
20484498 H.P.Su, Y.Yan, G.S.Prasad, R.F.Smith, C.L.Daniels, P.D.Abeywickrema, J.C.Reid, H.M.Loughran, M.Kornienko, S.Sharma, J.A.Grobler, B.Xu, V.Sardana, T.J.Allison, P.D.Williams, P.L.Darke, D.J.Hazuda, and S.Munshi (2010).
Structural basis for the inhibition of RNase H activity of HIV-1 reverse transcriptase by RNase H active site-directed inhibitors.
  J Virol, 84, 7625-7633.
PDB codes: 3lp0 3lp1 3lp2 3lp3
  20703329 J.E.Deweese, and N.Osheroff (2010).
The use of divalent metal ions by type II topoisomerases.
  Metallomics, 2, 450-459.  
20805464 M.Nadal, P.J.Mas, P.J.Mas, A.G.Blanco, C.Arnan, M.Solà, D.J.Hart, and M.Coll (2010).
Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain.
  Proc Natl Acad Sci U S A, 107, 16078-16083.
PDB codes: 3n4p 3n4q
21095591 M.P.Rychlik, H.Chon, S.M.Cerritelli, P.Klimek, R.J.Crouch, and M.Nowotny (2010).
Crystal structures of RNase H2 in complex with nucleic acid reveal the mechanism of RNA-DNA junction recognition and cleavage.
  Mol Cell, 40, 658-670.
PDB codes: 3o3f 3o3g 3o3h
20875084 N.Jongruja, D.J.You, E.Kanaya, Y.Koga, K.Takano, and S.Kanaya (2010).
The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity.
  FEBS J, 277, 4474-4489.  
19036793 E.Valkov, S.S.Gupta, S.Hare, A.Helander, P.Roversi, M.McClure, and P.Cherepanov (2009).
Functional and structural characterization of the integrase from the prototype foamy virus.
  Nucleic Acids Res, 37, 243-255.
PDB code: 3dlr
19161971 F.Xie, and C.M.Dupureur (2009).
Kinetic analysis of product release and metal ions in a metallonuclease.
  Arch Biochem Biophys, 483, 1-9.  
19119875 G.A.Cisneros, L.Perera, R.M.Schaaper, L.C.Pedersen, R.E.London, L.G.Pedersen, and T.A.Darden (2009).
Reaction mechanism of the epsilon subunit of E. coli DNA polymerase III: insights into active site metal coordination and catalytically significant residues.
  J Am Chem Soc, 131, 1550-1556.  
19647518 G.J.Grundy, S.Ramón-Maiques, E.K.Dimitriadis, S.Kotova, C.Biertümpfel, J.B.Heymann, A.C.Steven, M.Gellert, and W.Yang (2009).
Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex.
  Mol Cell, 35, 217-227.  
19228195 J.J.Champoux, and S.J.Schultz (2009).
Ribonuclease H: properties, substrate specificity and roles in retroviral reverse transcription.
  FEBS J, 276, 1506-1516.  
19490099 M.Jaskolski, J.N.Alexandratos, G.Bujacz, and A.Wlodawer (2009).
Piecing together the structure of retroviral integrase, an important target in AIDS therapy.
  FEBS J, 276, 2926-2946.  
19165139 M.Nowotny (2009).
Retroviral integrase superfamily: the structural perspective.
  EMBO Rep, 10, 144-151.  
19879839 N.D.Thomsen, and J.M.Berger (2009).
Running in reverse: the structural basis for translocation polarity in hexameric helicases.
  Cell, 139, 523-534.
PDB code: 3ice
19622742 S.E.Butcher (2009).
The spliceosome as ribozyme hypothesis takes a second step.
  Proc Natl Acad Sci U S A, 106, 12211-12212.  
19228196 S.M.Cerritelli, and R.J.Crouch (2009).
Ribonuclease H: the enzymes in eukaryotes.
  FEBS J, 276, 1494-1505.  
19228197 T.Tadokoro, and S.Kanaya (2009).
Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes.
  FEBS J, 276, 1482-1493.  
18836193 C.Dash, B.J.Scarth, C.Badorrek, M.Götte, and S.F.Le Grice (2008).
Examining the ribonuclease H primer grip of HIV-1 reverse transcriptase by charge neutralization of RNA/DNA hybrids.
  Nucleic Acids Res, 36, 6363-6371.  
18986998 J.Salon, J.Jiang, J.Sheng, O.O.Gerlits, and Z.Huang (2008).
Derivatization of DNAs with selenium at 6-position of guanine for function and crystal structure studies.
  Nucleic Acids Res, 36, 7009-7018.  
18662000 M.De Vivo, M.Dal Peraro, and M.L.Klein (2008).
Phosphodiester cleavage in ribonuclease H occurs via an associative two-metal-aided catalytic mechanism.
  J Am Chem Soc, 130, 10955-10962.  
18294720 M.L.Coté, and M.J.Roth (2008).
Murine leukemia virus reverse transcriptase: structural comparison with HIV-1 reverse transcriptase.
  Virus Res, 134, 186-202.  
18337749 M.Nowotny, S.M.Cerritelli, R.Ghirlando, S.A.Gaidamakov, R.J.Crouch, and W.Yang (2008).
Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD.
  EMBO J, 27, 1172-1181.
PDB code: 3bsu
18261820 S.J.Schultz, and J.J.Champoux (2008).
RNase H activity: structure, specificity, and function in reverse transcription.
  Virus Res, 134, 86.  
18408159 S.V.Lipchock, and S.A.Strobel (2008).
A relaxed active site after exon ligation by the group I intron.
  Proc Natl Acad Sci U S A, 105, 5699-5704.
PDB codes: 3bo2 3bo3 3bo4
18197661 U.D.Priyakumar, and A.D.Mackerell (2008).
Atomic detail investigation of the structure and dynamics of DNA.RNA hybrids: a molecular dynamics study.
  J Phys Chem B, 112, 1515-1524.  
18843295 V.Pena, A.Rozov, P.Fabrizio, R.Lührmann, and M.C.Wahl (2008).
Structure and function of an RNase H domain at the heart of the spliceosome.
  EMBO J, 27, 2929-2940.
PDB codes: 3e9l 3e9o 3e9p
18953336 W.Yang (2008).
An equivalent metal ion in one- and two-metal-ion catalysis.
  Nat Struct Mol Biol, 15, 1228-1231.  
  17374162 A.Savarino (2007).
In-Silico docking of HIV-1 integrase inhibitors reveals a novel drug type acting on an enzyme/DNA reaction intermediate.
  Retrovirology, 4, 21.  
17928262 C.R.Faehnle, and L.Joshua-Tor (2007).
Argonautes confront new small RNAs.
  Curr Opin Chem Biol, 11, 569-577.  
17189683 J.A.Worrall, and B.F.Luisi (2007).
Information available at cut rates: structure and mechanism of ribonucleases.
  Curr Opin Struct Biol, 17, 128-137.  
17637971 J.D.Ye, C.D.Barth, P.S.Anjaneyulu, T.Tuschl, and J.A.Piccirilli (2007).
Reactions of phosphate and phosphorothiolate diesters with nucleophiles: comparison of transition state structures.
  Org Biomol Chem, 5, 2491-2497.  
17214552 L.Mones, I.Simon, and M.Fuxreiter (2007).
Metal-binding sites at the active site of restriction endonuclease BamHI can conform to a one-ion mechanism.
  Biol Chem, 388, 73-78.  
17964265 M.Nowotny, S.A.Gaidamakov, R.Ghirlando, S.M.Cerritelli, R.J.Crouch, and W.Yang (2007).
Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription.
  Mol Cell, 28, 264-276.
PDB codes: 2qk9 2qkb 2qkk
  16880556 D.J.You, H.Chon, Y.Koga, K.Takano, and S.Kanaya (2006).
Crystallization and preliminary crystallographic analysis of type 1 RNase H from the hyperthermophilic archaeon Sulfolobus tokodaii 7.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 62, 781-784.  
17029813 J.S.Parker, and D.Barford (2006).
Argonaute: A scaffold for the function of short regulatory RNAs.
  Trends Biochem Sci, 31, 622-630.  
17085478 T.L.Diamond, and F.D.Bushman (2006).
Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay.
  Nucleic Acids Res, 34, 6116-6125.  
16600865 W.Yang, J.Y.Lee, and M.Nowotny (2006).
Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity.
  Mol Cell, 22, 5.  
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.