PDBsum entry 2be5

Transferase

PDB id: 2be5

Contents

Protein chains

229 a.a. *

1119 a.a. *

1392 a.a. *

95 a.a. *

345 a.a. *

Ligands

TGT ×2

Metals

_MG ×4

_ZN ×4

Waters ×8184

* Residue conservation analysis

PDB id:

2be5

Name:

Transferase

Title:

Crystal structure of the t. Thermophilus RNA polymerase holoenzyme in complex with inhibitor tagetitoxin

Structure:

DNA-directed RNA polymerase alpha chain. Chain: a, b, k, l. Synonym: rnap alpha subunit, transcriptase alpha chain, RNA polymerase alpha subunit. DNA-directed RNA polymerase beta chain. Chain: c, m. Synonym: rnap beta subunit, transcriptase beta chain, RNA polymerase beta subunit. DNA-directed RNA polymerase beta' chain.

Source:

Thermus thermophilus. Organism_taxid: 274. Organism_taxid: 274

Biol. unit:

Hexamer (from PQS)

Resolution:

2.40Å R-factor: 0.237 R-free: 0.274

Authors:

D.G.Vassylyev,V.Svetlov,M.N.Vassylyeva,A.Perederina,N.Igarashi, N.Matsugaki,S.Wakatsuki,I.Artsimovitch,Riken Structural Genomics/proteomics Initiative (Rsgi)

Key ref:

D.G.Vassylyev et al. (2005). Structural basis for transcription inhibition by tagetitoxin. Nat Struct Mol Biol, 12, 1086-1093. PubMed id: 16273103 DOI: 10.1038/nsmb1015

Date:

22-Oct-05 Release date: 08-Nov-05

Protein chains

Q5SHR6  (RPOA_THET8) -  DNA-directed RNA polymerase subunit alpha from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 315 a.a.

Struc: 229 a.a.

Protein chains

Q8RQE9  (RPOB_THET8) -  DNA-directed RNA polymerase subunit beta from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 1119 a.a.

Struc: 1119 a.a.

Protein chains

Q8RQE8  (RPOC_THET8) -  DNA-directed RNA polymerase subunit beta' from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 1524 a.a.

Struc: 1392 a.a.

Protein chains

Q8RQE7  (RPOZ_THET8) -  DNA-directed RNA polymerase subunit omega from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 99 a.a.

Struc: 95 a.a.*

Protein chains

Q5SKW1  (Q5SKW1_THET8) -  RNA polymerase sigma factor SigA from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 423 a.a.

Struc: 345 a.a.

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B, C, D, E, K, L, M, N, O: 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: 10.1038/nsmb1015

Nat Struct Mol Biol 12:1086-1093 (2005)

PubMed id: 16273103

Structural basis for transcription inhibition by tagetitoxin.

D.G.Vassylyev, V.Svetlov, M.N.Vassylyeva, A.Perederina, N.Igarashi, N.Matsugaki, S.Wakatsuki, I.Artsimovitch.

ABSTRACT

Tagetitoxin (Tgt) inhibits transcription by an unknown mechanism. A structure at a resolution of 2.4 A of the Thermus thermophilus RNA polymerase (RNAP)-Tgt complex revealed that the Tgt-binding site within the RNAP secondary channel overlaps that of the stringent control effector ppGpp, which partially protects RNAP from Tgt inhibition. Tgt binding is mediated exclusively through polar interactions with the beta and beta' residues whose substitutions confer resistance to Tgt in vitro. Importantly, a Tgt phosphate, together with two active site acidic residues, coordinates the third Mg(2+) ion, which is distinct from the two catalytic metal ions. We show that Tgt inhibits all RNAP catalytic reactions and propose a mechanism in which the Tgt-bound Mg(2+) ion has a key role in stabilization of an inactive transcription intermediate. Remodeling of the active site by metal ions could be a common theme in the regulation of catalysis by nucleic acid enzymes.

Selected figure(s)

Figure 3.
Figure 3. Tgt and ppGpp bind to overlapping sites on RNAP. (a) Superposition of the ttRNAP-Tgt and ttRNAP-ppGpp complexes. The color scheme is the same as in Figures 1a and 2a. (b,c) Structural determinants probably crucial for Tgt and ppGpp binding. The RNAP-Tgt (b) and RNAP-ppGpp (c) complexes are shown in the same orientation for better comparison. Residues that are not identical between E. coli and T. thermophilus are marked by their numbers only. RNAP residues (balls and sticks) that interact with both Tgt and ppGpp are shown in green, whereas those specific for Tgt and ppGpp are shown in light cyan and light pink, respectively. (d) ppGpp and DksA compete with Tgt for the inhibition of abortive transcription by the wild-type ecRNAP from the T7A1 promoter. The assay was conducted as in Figure 2d. ppGpp was added to 0.5 mM, DksA to 500 nM.

Figure 5.
Figure 5. Mechanism of Tgt action. (a-c) Models of the substrates (light green) corresponding to the three postulated consecutive steps during NTP loading to the RNAP active site are superimposed on the RNAP-Tgt structure; the E site (a), the preinsertion site (b) and the insertion site (c); the PDB accession codes used in panels a, b and c were 1R9T, 1Y77 and 1R9S, respectively. The putative coordination bonds with tMG and/or cMG2 of Tgt (cyan), the NTP in the preinsertion site and the -phosphate (P ) of the NTP in the insertion site (light green) (b,c), as well as of the NTP -phosphates in the insertion site (c) in the 'catalytic' cP (yellow) and 'inactive', tMG-bound tP (red) configurations are shown by dashed lines. (d) Stabilization of an inactive transcription intermediate by Tgt.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2005, 12, 1086-1093) copyright 2005.

Figures were selected by the author.