PDBsum entry 2pi5

Transferase/DNA

PDB id: 2pi5

Contents

Protein chain

862 a.a. *

DNA/RNA

* Residue conservation analysis

PDB id:

2pi5

Name:

Transferase/DNA

Title:

T7 RNA polymerase complexed with a phi10 promoter

Structure:

5'-d( Cp Tp Tp C 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. DNA-directed RNA polymerase. Chain: a.

Source:

Synthetic: yes. Enterobacteria phage t7. Organism_taxid: 10760. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.90Å R-factor: 0.249 R-free: 0.268

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

Protein chain

P00573  (RPOL_BPT7) -  T7 RNA polymerase from Escherichia phage T7

Seq: 883 a.a.

Struc: 862 a.a.

DNA/RNA chains

C-T-A-T-A-G-T-G-A-G-T-C-G-T-A-T-T-A 18 bases

T-A-A-T-A-C-G-A-C-T-C-A-C-T 14 bases

Enzyme reactions

• Enzyme class: E.C.2.7.7.6 - DNA-directed Rna polymerase.
• Reaction: RNA(n) + a ribonucleoside 5'-triphosphate = RNA(n+1) + diphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added 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.