PDBsum entry 3eql

Transferase

PDB id: 3eql

Contents

Protein chains

229 a.a. *

1119 a.a. *

1321 a.a. *

95 a.a. *

345 a.a. *

Ligands

MXP ×2

Metals

_MG ×2

_ZN ×4

Waters ×4496

* Residue conservation analysis

PDB id:

3eql

Name:

Transferase

Title:

Crystal structure of the t. Thermophilus RNA polymerase holoenzyme in complex with antibiotic myxopyronin

Structure:

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

Source:

Thermus thermophilus. Organism_taxid: 274. Organism_taxid: 274

Resolution:

2.70Å R-factor: 0.240 R-free: 0.270

Authors:

D.G.Vassylyev,M.N.Vassylyeva,I.Artsimovitch

Key ref:

G.A.Belogurov et al. (2009). Transcription inactivation through local refolding of the RNA polymerase structure. Nature, 457, 332-335. PubMed id: 18946472 DOI: 10.1038/nature07510

Date:

30-Sep-08 Release date: 28-Oct-08

Protein chains

Q9Z9H6  (RPOA_THETH) -  DNA-directed RNA polymerase subunit alpha from Thermus thermophilus

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: 1321 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.

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/nature07510

Nature 457:332-335 (2009)

PubMed id: 18946472

Transcription inactivation through local refolding of the RNA polymerase structure.

G.A.Belogurov, M.N.Vassylyeva, A.Sevostyanova, J.R.Appleman, A.X.Xiang, R.Lira, S.E.Webber, S.Klyuyev, E.Nudler, I.Artsimovitch, D.G.Vassylyev.

ABSTRACT

Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx-a desmethyl derivative of myxopyronin B-complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the beta'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex-the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.

Selected figure(s)

Figure 3.
Figure 3: A mechanism of the dMyx action. a, Myx alters the contacts between RNAP and P[R] promoter DNA. A linear DNA fragment encompassing positions -81 to +70 of the P[R] promoter was generated by polymerase chain reaction (PCR); the non-template DNA strand was end-labelled with [^32P]- ATP (see Methods). The sequence from -44 to +23 is shown. The -35 and -10 hexamers are indicated by black boxes, the start site (+1) is shown by a black dot. The top panel shows probing of the non-template strand by piperidine-induced cleavage of the permanganate-modified T residues. Reactivities of -10, -4, -3 and +2 residues (quantification described in Methods) are shown to the left of the gel and summarized above the promoter sequence where black and white arrows indicate high and low reactivity, respectively. The bottom panel shows protection of the non-template DNA strand from DNaseI digestion. The footprint boundaries in the promoter region shown are indicated on the gel and by black (RNAP alone) and white (RNAP with the inhibitor) bars below the promoter sequence; the dideoxy-sequencing ladder is shown for reference. In the gels shown, independent reaction repeats were analysed for consistency. b, Schematic drawing of the putative mechanism of the dMyx action. dwDNA, downstream DNA.

Figure 4.
Figure 4: Mutations in switch-2 affect the open complex formation. Accessibility of the non-template DNA strand residues to permanganate modification probed as in Fig. 3a. Wild-type and mutant RNAPs differ in their patterns of reactivity in the absence of dMyx (top traces) but are nearly identical in the presence of 10 M dMyx (bottom traces). Notably, ' 309–325 that removes the entire rudder loop (which is inserted in the same helix as switch-2, but is unlikely to interfere with the nucleic acids) has no effect on DNA melting, suggesting that a melting defect of a different rudder deletion^19 might be due to changes in the adjacent switch-2 instead.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: Nature (2009, 457, 332-335) copyright 2009.

Figures were selected by an automated process.