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PDBsum entry 3cqz

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protein ligands metals Protein-protein interface(s) links
Transcription/toxin PDB id
3cqz
Jmol
Contents
Protein chains
1349 a.a. *
1061 a.a. *
265 a.a. *
213 a.a. *
84 a.a. *
116 a.a. *
121 a.a. *
65 a.a. *
113 a.a. *
43 a.a. *
Ligands
ILX-TRX-GLY-ILE-
GLY-CSX-ASN-HYP
Metals
_ZN ×8
Waters ×17
* Residue conservation analysis
PDB id:
3cqz
Name: Transcription/toxin
Title: Crystal structure of 10 subunit RNA polymerase ii in complex inhibitor alpha-amanitin
Structure: DNA-directed RNA polymerase ii subunit rpb1. Chain: a. Synonym: RNA polymerase ii subunit b1, RNA polymerase ii su DNA-directed RNA polymerase iii largest subunit, b220. DNA-directed RNA polymerase ii subunit rpb2. Chain: b. Synonym: RNA polymerase ii subunit 2, DNA-directed RNA poly 140 kda polypeptide, b150. DNA-directed RNA polymerase ii subunit rpb3.
Source: Saccharomyces cerevisiae. Brewer's yeast, lager beer yeast, yeast. Organism_taxid: 4932. Synthetic: yes. Amanita phalloides. Organism_taxid: 67723
Resolution:
2.80Å     R-factor:   0.202     R-free:   0.273
Authors: C.D.Kaplan,K.-M.Larsson,R.D.Kornberg
Key ref:
C.D.Kaplan et al. (2008). The RNA polymerase II trigger loop functions in substrate selection and is directly targeted by alpha-amanitin. Mol Cell, 30, 547-556. PubMed id: 18538653 DOI: 10.1016/j.molcel.2008.04.023
Date:
03-Apr-08     Release date:   22-Jul-08    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P04050  (RPB1_YEAST) -  DNA-directed RNA polymerase II subunit RPB1
Seq:
Struc:
 
Seq:
Struc:
 
Seq:
Struc:
 
Seq:
Struc:
1733 a.a.
1349 a.a.
Protein chain
Pfam   ArchSchema ?
P08518  (RPB2_YEAST) -  DNA-directed RNA polymerase II subunit RPB2
Seq:
Struc:
 
Seq:
Struc:
 
Seq:
Struc:
1224 a.a.
1061 a.a.
Protein chain
Pfam   ArchSchema ?
P16370  (RPB3_YEAST) -  DNA-directed RNA polymerase II subunit RPB3
Seq:
Struc:
318 a.a.
265 a.a.
Protein chain
Pfam   ArchSchema ?
P20434  (RPAB1_YEAST) -  DNA-directed RNA polymerases I, II, and III subunit RPABC1
Seq:
Struc:
215 a.a.
213 a.a.
Protein chain
Pfam   ArchSchema ?
P20435  (RPAB2_YEAST) -  DNA-directed RNA polymerases I, II, and III subunit RPABC2
Seq:
Struc:
155 a.a.
84 a.a.
Protein chain
Pfam   ArchSchema ?
P20436  (RPAB3_YEAST) -  DNA-directed RNA polymerases I, II, and III subunit RPABC3
Seq:
Struc:
146 a.a.
116 a.a.
Protein chain
Pfam   ArchSchema ?
P27999  (RPB9_YEAST) -  DNA-directed RNA polymerase II subunit RPB9
Seq:
Struc:
122 a.a.
121 a.a.
Protein chain
Pfam   ArchSchema ?
P22139  (RPAB5_YEAST) -  DNA-directed RNA polymerases I, II, and III subunit RPABC5
Seq:
Struc:
70 a.a.
65 a.a.
Protein chain
Pfam   ArchSchema ?
P38902  (RPB11_YEAST) -  DNA-directed RNA polymerase II subunit RPB11
Seq:
Struc:
120 a.a.
113 a.a.
Protein chain
Pfam   ArchSchema ?
P40422  (RPAB4_YEAST) -  DNA-directed RNA polymerases I, II, and III subunit RPABC4
Seq:
Struc:
70 a.a.
43 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: Chains A, B: 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
+ 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!
  Cellular component     cytoplasm   7 terms 
  Biological process     transcription, RNA-dependent   14 terms 
  Biochemical function     RNA polymerase II activity     14 terms  

 

 
    reference    
 
 
DOI no: 10.1016/j.molcel.2008.04.023 Mol Cell 30:547-556 (2008)
PubMed id: 18538653  
 
 
The RNA polymerase II trigger loop functions in substrate selection and is directly targeted by alpha-amanitin.
C.D.Kaplan, K.M.Larsson, R.D.Kornberg.
 
  ABSTRACT  
 
Structural, biochemical, and genetic studies have led to proposals that a mobile element of multisubunit RNA polymerases, the Trigger Loop (TL), plays a critical role in catalysis and can be targeted by antibiotic inhibitors. Here we present evidence that the Saccharomyces cerevisiae RNA Polymerase II (Pol II) TL participates in substrate selection. Amino acid substitutions within the Pol II TL preferentially alter substrate usage and enzyme fidelity, as does inhibition of transcription by alpha-amanitin. Finally, substitution of His1085 in the TL specifically renders Pol II highly resistant to alpha-amanitin, indicating a functional interaction between His1085 and alpha-amanitin that is supported by rerefinement of an alpha-amanitin-Pol II crystal structure. We propose that alpha-amanitin-inhibited Pol II elongation, which is slow and exhibits reduced substrate selectivity, results from direct alpha-amanitin interference with the TL.
 
  Selected figure(s)  
 
Figure 2.
Figure 2. Elongation Defects and Altered Substrate Selection by Rpb1 H1085Y Pol II
(A) H1085Y exhibits reduced elongation rate using NTP substrates. Run-off transcription of an oligonucleotide scaffold template generates a 61 nt RNA product. Representative experiments for WT and H1085Y Pol II are shown in the left and right panels, respectively. Average elongation rates for each NTP concentration were measured as the length of the transcribed region (51 nt) divided by the time of half-maximal accumulation of run-off product (61 nt). Average elongation rates were then plotted versus NTP concentration to infer maximum average elongation rate (see Experimental Procedures for details) (top right graph). Inferred maximum average elongation rates are shown in the bottom right graph with error bars representing the 95% confidence interval (See Experimental Procedures for details).
(B) H1085Y Pol II exhibits only modest defects for 2′-dNTP incorporation. WT and H1085Y Pol II ECs were formed on oligonucleotide scaffolds containing 10-mer RNAs with templates specifying addition of different NTPs at position 11. Average incorporation rates for different template-specified 2′-dNTPs were measured as 1/t[1/2] for maximal accumulation of 11-mer RNA. Incorporation rates were then plotted versus 2′-dNTP concentration, and maximum incorporation rate for either 2′-dATP or 2′-dGTP was inferred (left panels). Maximum incorporation rates for WT Pol II and H1085Y are shown in the right panels with error bars representing the 95% confidence interval (See Experimental Procedures for details).
(C) H1085Y Pol II exhibits modest defects in GTP misincorporation. WT and H1085Y ECs were formed and labeled as in (B) with templates specifying incorporation of ATP at the position being measured, but were challenged with 1 mM GTP and misincorporation rate measured as the 1/t[1/2] for maximal incorporation. Mean misincorporation rate from at least three experiments is represented in the bar graph (error bars represent ± SD).
Figure 7.
Figure 7. Direct Interaction between Rpb1 His1085 and α-Amanitin and TL Capture May Underlie α-Amanitin Inhibition of Transcription
(A) Overall view of α-amanitin and the new TL conformation and their positions in relation to the Bridge helix (BH). A superpositioned EC structure (PDB 2E2H) showing DNA (magenta), RNA (red), nontemplate DNA (green), and nucleotide GTP (orange) highlights the position of the inhibitor and TL in relation to EC components.
(B) A 90° rotation shows the α-amanitin position in relation to the Bridge helix (BH) and its capture of the TL Rpb1 His1085.
(C) TL residues Rpb1 1084–1086 and the entire α-amanitin modeled into electron density (dark gray mesh) from an initial unbiased 2Fo-Fc electron density map contoured at 0.6 σ.
 
  The above figures are reprinted from an Open Access publication published by Cell Press: Mol Cell (2008, 30, 547-556) copyright 2008.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21292158 M.H.Larson, R.Landick, and S.M.Block (2011).
Single-molecule studies of RNA polymerase: one singular sensation, every little step it takes.
  Mol Cell, 41, 249-262.  
21447716 S.R.Kennedy, and D.A.Erie (2011).
Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription.
  Proc Natl Acad Sci U S A, 108, 6079-6084.  
20598112 C.D.Kaplan (2010).
The architecture of RNA polymerase fidelity.
  BMC Biol, 8, 85.  
20088966 H.Koyama, T.Ueda, T.Ito, and K.Sekimizu (2010).
Novel RNA polymerase II mutation suppresses transcriptional fidelity and oxidative stress sensitivity in rpb9Delta yeast.
  Genes Cells, 15, 151-159.  
19575656 J.B.Hollick (2010).
Paramutation and development.
  Annu Rev Cell Dev Biol, 26, 557-579.  
19966797 J.Zhang, M.Palangat, and R.Landick (2010).
Role of the RNA polymerase trigger loop in catalysis and pausing.
  Nat Struct Mol Biol, 17, 99.  
20367031 L.A.Selth, S.Sigurdsson, and J.Q.Svejstrup (2010).
Transcript Elongation by RNA Polymerase II.
  Annu Rev Biochem, 79, 271-293.  
  21326898 N.Miropolskaya, V.Nikiforov, S.Klimašauskas, I.Artsimovitch, and A.Kulbachinskiy (2010).
Modulation of RNA polymerase activity through trigger loop folding.
  Transcr, 1, 89-94.  
  20856905 N.Opalka, J.Brown, W.J.Lane, K.A.Twist, R.Landick, F.J.Asturias, and S.A.Darst (2010).
Complete structural model of Escherichia coli RNA polymerase from a hybrid approach.
  PLoS Biol, 8, 0.
PDB codes: 3lti 3lu0
21034443 R.O.Weinzierl (2010).
The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain.
  BMC Biol, 8, 134.  
19895816 W.J.Lane, and S.A.Darst (2010).
Molecular evolution of multisubunit RNA polymerases: structural analysis.
  J Mol Biol, 395, 686-704.  
19895820 W.J.Lane, and S.A.Darst (2010).
Molecular evolution of multisubunit RNA polymerases: sequence analysis.
  J Mol Biol, 395, 671-685.  
20798057 X.Huang, D.Wang, D.R.Weiss, D.A.Bushnell, R.D.Kornberg, and M.Levitt (2010).
RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription.
  Proc Natl Acad Sci U S A, 107, 15745-15750.  
20459653 Y.Yuzenkova, A.Bochkareva, V.R.Tadigotla, M.Roghanian, S.Zorov, K.Severinov, and N.Zenkin (2010).
Stepwise mechanism for transcription fidelity.
  BMC Biol, 8, 54.  
20534498 Y.Yuzenkova, and N.Zenkin (2010).
Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis.
  Proc Natl Acad Sci U S A, 107, 10878-10883.  
19151724 C.Castro, E.D.Smidansky, J.J.Arnold, K.R.Maksimchuk, I.Moustafa, A.Uchida, M.Götte, W.Konigsberg, and C.E.Cameron (2009).
Nucleic acid polymerases use a general acid for nucleotidyl transfer.
  Nat Struct Mol Biol, 16, 212-218.  
19910183 C.E.Cameron, I.M.Moustafa, and J.J.Arnold (2009).
Dynamics: the missing link between structure and function of the viral RNA-dependent RNA polymerase?
  Curr Opin Struct Biol, 19, 768-774.  
19451224 C.Mosrin-Huaman, R.Honorine, and A.R.Rahmouni (2009).
Expression of bacterial Rho factor in yeast identifies new factors involved in the functional interplay between transcription and mRNP biogenesis.
  Mol Cell Biol, 29, 4033-4044.  
19439405 C.Walmacq, M.L.Kireeva, J.Irvin, Y.Nedialkov, L.Lubkowska, F.Malagon, J.N.Strathern, and M.Kashlev (2009).
Rpb9 Subunit Controls Transcription Fidelity by Delaying NTP Sequestration in RNA Polymerase II.
  J Biol Chem, 284, 19601-19612.  
19478184 D.Wang, D.A.Bushnell, X.Huang, K.D.Westover, M.Levitt, and R.D.Kornberg (2009).
Structural basis of transcription: backtracked RNA polymerase II at 3.4 angstrom resolution.
  Science, 324, 1203-1206.
PDB codes: 3gtg 3gtj 3gtk 3gtl 3gtm 3gto 3gtp 3gtq
19489723 E.Nudler (2009).
RNA polymerase active center: the molecular engine of transcription.
  Annu Rev Biochem, 78, 335-361.  
19171965 F.Brueckner, K.J.Armache, A.Cheung, G.E.Damsma, H.Kettenberger, E.Lehmann, J.Sydow, and P.Cramer (2009).
Structure-function studies of the RNA polymerase II elongation complex.
  Acta Crystallogr D Biol Crystallogr, 65, 112-120.  
19458260 H.Spåhr, G.Calero, D.A.Bushnell, and R.D.Kornberg (2009).
Schizosacharomyces pombe RNA polymerase II at 3.6-A resolution.
  Proc Natl Acad Sci U S A, 106, 9185-9190.
PDB code: 3h0g
19560423 J.F.Sydow, F.Brueckner, A.C.Cheung, G.E.Damsma, S.Dengl, E.Lehmann, D.Vassylyev, and P.Cramer (2009).
Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA.
  Mol Cell, 34, 710-721.
PDB codes: 3hou 3hov 3how 3hox 3hoy 3hoz
19251626 K.F.Erhard, J.L.Stonaker, S.E.Parkinson, J.P.Lim, C.J.Hale, and J.B.Hollick (2009).
RNA polymerase IV functions in paramutation in Zea mays.
  Science, 323, 1201-1205.  
19416863 M.L.Kireeva, and M.Kashlev (2009).
Mechanism of sequence-specific pausing of bacterial RNA polymerase.
  Proc Natl Acad Sci U S A, 106, 8900-8905.  
19855007 N.Miropolskaya, I.Artsimovitch, S.Klimasauskas, V.Nikiforov, and A.Kulbachinskiy (2009).
Allosteric control of catalysis by the F loop of RNA polymerase.
  Proc Natl Acad Sci U S A, 106, 18942-18947.  
19055851 L.Tan, S.Wiesler, D.Trzaska, H.C.Carney, and R.O.Weinzierl (2008).
Bridge helix and trigger loop perturbations generate superactive RNA polymerases.
  J Biol, 7, 40.  
18957193 R.Sousa (2008).
Tie me up, tie me down: inhibiting RNA polymerase.
  Cell, 135, 205-207.  
18679430 V.Svetlov, and E.Nudler (2008).
Jamming the ratchet of transcription.
  Nat Struct Mol Biol, 15, 777-779.  
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.