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PDBsum entry 2f23
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Transcription
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PDB id
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2f23
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References listed in PDB file
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Key reference
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Title
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Ph-Dependent conformational switch activates the inhibitor of transcription elongation.
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Authors
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O.Laptenko,
S.S.Kim,
J.Lee,
M.Starodubtseva,
F.Cava,
J.Berenguer,
X.P.Kong,
S.Borukhov.
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Ref.
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EMBO J, 2006,
25,
2131-2141.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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.
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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).
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
EMBO J
(2006,
25,
2131-2141)
copyright 2006.
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