PDBsum entry 2f23

Transcription

PDB id: 2f23

Contents

Protein chains

154 a.a. *

Waters ×292

* Residue conservation analysis

PDB id:

2f23

Name:

Transcription

Title:

Crystal structure of grea factor homolog 1 (gfh1) protein of thermus thermophilus

Structure:

Anti-cleavage anti-grea transcription factor gfh1. Chain: a, b. Engineered: yes

Source:

Thermus thermophilus. Organism_taxid: 262724. Strain: hb27. Gene: gfh1. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Resolution:

1.60Å R-factor: 0.206 R-free: 0.254

Authors:

X.P.Kong,S.-S.Kim

Key ref:

O.Laptenko et al. (2006). pH-dependent conformational switch activates the inhibitor of transcription elongation. EMBO J, 25, 2131-2141. PubMed id: 16628221 DOI: 10.1038/sj.emboj.7601094

Date:

15-Nov-05 Release date: 30-May-06

Protein chains

Q72JT8  (GFH1_THET2) -  Transcription inhibitor protein Gfh1 from Thermus thermophilus (strain ATCC BAA-163 / DSM 7039 / HB27)

Seq: 156 a.a.

Struc: 154 a.a.

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1038/sj.emboj.7601094

EMBO J 25:2131-2141 (2006)

PubMed id: 16628221

pH-dependent conformational switch activates the inhibitor of transcription elongation.

O.Laptenko, S.S.Kim, J.Lee, M.Starodubtseva, F.Cava, J.Berenguer, X.P.Kong, S.Borukhov.

ABSTRACT

Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase (RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N-terminal coiled-coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C-terminal domain (CTD). We show that depending on pH, Gfh1-CTD exists in two alternative orientations. At pH above 7, it assumes an inactive 'flipped' orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1-CTD switches to an active 'Gre-like' orientation, which enables Gfh1 to bind to and inhibit RNAP.

Selected figure(s)

Figure 5.
Figure 5 Crystal structure of Tth Gfh1. (A) Ribbon representation of Gfh1 structure is shown in two orthogonal views (left and central panel). The location of the two domains, NTD and CTD, and the four Asp residues of NTD loop are indicated. The structure of E. coli GreA (Stebbins et al, 1995) is shown for comparison (right panel). (B) Superposition of the structures of E. coli GreA (orange) and Tth Gfh1 (blue) as C[ ]trace. Left panel shows the alignment by NTD and central panel shows alignment by CTD. Overall, the molecules superimposed well with r.m.s. deviation of the C[ ]atoms=1.8 Å with more than 90% equivalences. The rotation axis of the CTD is indicated. Right panel shows aligned NTDs, rotated by 60° counterclockwise around the NTD axis from the view shown in the left panel.

Figure 6.
Figure 6 Effect of CTD conformation on functional activity of Gfh1. (A) Model structures of mutant factors with conformations fixed via S–S bridges, Gfh1-CC12 and Gfh1-CC13 are shown as ribbons. Models were generated by Swiss-Model (Schwede et al, 2003) using the structures of Tth Gfh1 and E. coli GreA as templates, respectively. (B) Summary of the inhibitory activities of wt and mutant Gfh1-CC factors. The IC[50] values were obtained from abortive initiation assay as in Figure 4A–C, conducted under indicated conditions. (C) [^32P]Gfh1–RNAP competition-binding assay. [^32P]Gfh1–RNAP core complex was chromatographed with or without 20 M competitor proteins, Gfh1-CC12 or Gfh1-CC13, at pH 6.4 under nonreducing conditions (see Figure 4D). Free oxidized forms of Gfh1-CC12 and Gfh1-CC13 all elute irrespective of pH with almost identical retention times of 24.5–24.7 min (the same as that of the wt Gfh1) corresponding to an apparent molecular weight of 26 kDa (data not shown), which, according to a light-scattering analysis, represents a monomer (see Supplementary data).

The above figures are reprinted by permission from Macmillan Publishers Ltd: EMBO J (2006, 25, 2131-2141) copyright 2006.

Figures were selected by the author.