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PDBsum entry 2pi4

Go to PDB code: 
protein dna_rna ligands metals links
Transferase/DNA PDB id
2pi4
Jmol
Contents
Protein chain
862 a.a. *
DNA/RNA
Ligands
GH3 ×2
Metals
_MG ×2
Waters ×129
* Residue conservation analysis
PDB id:
2pi4
Name: Transferase/DNA
Title: T7rnap complexed with a phi10 protein and initiating gtps.
Structure: 5'- d( Cp Tp Tp Cp Cp Tp Ap Tp Ap Gp Tp Gp Ap Gp Tp Cp Gp Tp Ap Tp Tp A)-3'. Chain: t. Engineered: yes. 5'-d( Tp Ap Ap Tp Ap Cp Gp Ap Cp Tp Cp Ap Cp T)- 3'. Chain: p. Engineered: yes.
Source: Synthetic: yes. Enterobacteria phage t7. Organism_taxid: 10760. Gene: 1. Expressed in: escherichia coli. Expression_system_taxid: 562
Resolution:
2.50Å     R-factor:   0.265     R-free:   0.297
Authors: W.P.Kennedy,J.R.Momand,Y.W.Yin
Key ref:
W.P.Kennedy et al. (2007). Mechanism for de novo RNA synthesis and initiating nucleotide specificity by t7 RNA polymerase. J Mol Biol, 370, 256-268. PubMed id: 17512007 DOI: 10.1016/j.jmb.2007.03.041
Date:
12-Apr-07     Release date:   19-Jun-07    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P00573  (RPOL_BPT7) -  T7 RNA polymerase
Seq:
Struc:
 
Seq:
Struc:
883 a.a.
862 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.2.7.7.6  - DNA-directed Rna polymerase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Nucleoside triphosphate + RNA(n) = diphosphate + RNA(n+1)
Nucleoside triphosphate
Bound ligand (Het Group name = GH3)
matches with 60.00% similarity
+ RNA(n)
= diphosphate
+ RNA(n+1)
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     transcription, DNA-dependent   1 term 
  Biochemical function     transferase activity     4 terms  

 

 
    reference    
 
 
DOI no: 10.1016/j.jmb.2007.03.041 J Mol Biol 370:256-268 (2007)
PubMed id: 17512007  
 
 
Mechanism for de novo RNA synthesis and initiating nucleotide specificity by t7 RNA polymerase.
W.P.Kennedy, J.R.Momand, Y.W.Yin.
 
  ABSTRACT  
 
DNA-directed RNA polymerases are capable of initiating synthesis of RNA without primers, the first catalytic stage of initiation is referred to as de novo RNA synthesis. De novo synthesis is a unique phase in the transcription cycle where the RNA polymerase binds two nucleotides rather than a nascent RNA polymer and a single nucleotide. For bacteriophage T7 RNA polymerase, transcription begins with a marked preference for GTP at the +1 and +2 positions. We determined the crystal structures of T7 RNA polymerase complexes captured during the de novo RNA synthesis. The DNA substrates in the structures in the complexes contain a common Phi10 duplex promoter followed by a unique five base single-stranded extension of template DNA whose sequences varied at positions +1 and +2, thereby allowing for different pairs of initiating nucleotides GTP, ATP, CTP or UTP to bind. The structures show that the initiating nucleotides bind RNA polymerase in locations distinct from those described previously for elongation complexes. Selection bias in favor of GTP as an initiating nucleotide is accomplished by shape complementarity, extensive protein side-chain and strong base-stacking interactions for the guanine moiety in the enzyme active site. Consequently, an initiating GTP provides the largest stabilization force for the open promoter conformation.
 
  Selected figure(s)  
 
Figure 1.
Figure 1. Configuration of the active site of T7 RNAP before and after NTP binding. (a) The pre-insertion conformation that is incompetent for NTP binding. The templating residue n is in a flipped-out position; the NTP-binding site (N-site) is occluded by Y639 and the O-helix is inward, away from the NTP site. (b) The post insertion conformation induced by NTP binding. The conformational changes move Y639 away from the N-site, repositions the templating residue n, and rotates the O-helix towards the active site.
Figure 4.
Figure 4. Guanine-specific interactions between the initiating GTP nucleotides and the active site residues of the polymerase. (a) Interaction of the first G:C base-pair between GTP (gold) and the + 1 cytosine on the template (blue) with RNAP. (b) Interaction of the second G:C base-pair between GTP (gold) and the + 2 cytosine (blue) on the template with the RNAP.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 370, 256-268) copyright 2007.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21321236 M.L.Gleghorn, E.K.Davydova, R.Basu, L.B.Rothman-Denes, and K.S.Murakami (2011).
X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides.
  Proc Natl Acad Sci U S A, 108, 3566-3571.
PDB codes: 3q0a 3q22 3q23 3q24
20129056 J.M.Fusté, S.Wanrooij, E.Jemt, C.E.Granycome, T.J.Cluett, Y.Shi, N.Atanassova, I.J.Holt, C.M.Gustafsson, and M.Falkenberg (2010).
Mitochondrial RNA polymerase is needed for activation of the origin of light-strand DNA replication.
  Mol Cell, 37, 67-78.  
20008320 S.Paratkar, and S.S.Patel (2010).
Mitochondrial transcription factor Mtf1 traps the unwound non-template strand to facilitate open complex formation.
  J Biol Chem, 285, 3949-3956.  
19015264 E.K.Davydova, I.Kaganman, K.M.Kazmierczak, and L.B.Rothman-Denes (2009).
Identification of bacteriophage n4 virion RNA polymerase-nucleic Acid interactions in transcription complexes.
  J Biol Chem, 284, 1962-1970.  
19220054 J.N.Pitt, and A.R.Ferré-D'Amaré (2009).
Structure-guided engineering of the regioselectivity of RNA ligase ribozymes.
  J Am Chem Soc, 131, 3532-3540.
PDB codes: 3fs0 3ftm
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