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PDBsum entry 3cqz
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Transcription/toxin
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PDB id
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3cqz
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Contents |
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1349 a.a.
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1061 a.a.
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265 a.a.
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213 a.a.
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84 a.a.
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116 a.a.
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121 a.a.
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65 a.a.
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113 a.a.
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43 a.a.
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References listed in PDB file
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Key reference
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Title
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The RNA polymerase ii trigger loop functions in substrate selection and is directly targeted by alpha-Amanitin.
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Authors
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C.D.Kaplan,
K.M.Larsson,
R.D.Kornberg.
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Ref.
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Mol Cell, 2008,
30,
547-556.
[DOI no: ]
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PubMed id
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Abstract
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Structural, biochemical, and genetic studies have led to proposals that a mobile
element of multisubunit RNA polymerases, the Trigger Loop (TL), plays a critical
role in catalysis and can be targeted by antibiotic inhibitors. Here we present
evidence that the Saccharomyces cerevisiae RNA Polymerase II (Pol II) TL
participates in substrate selection. Amino acid substitutions within the Pol II
TL preferentially alter substrate usage and enzyme fidelity, as does inhibition
of transcription by alpha-amanitin. Finally, substitution of His1085 in the TL
specifically renders Pol II highly resistant to alpha-amanitin, indicating a
functional interaction between His1085 and alpha-amanitin that is supported by
rerefinement of an alpha-amanitin-Pol II crystal structure. We propose that
alpha-amanitin-inhibited Pol II elongation, which is slow and exhibits reduced
substrate selectivity, results from direct alpha-amanitin interference with the
TL.
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Figure 2.
Figure 2. Elongation Defects and Altered Substrate Selection
by Rpb1 H1085Y Pol II
(A) H1085Y exhibits reduced
elongation rate using NTP substrates. Run-off transcription of
an oligonucleotide scaffold template generates a 61 nt RNA
product. Representative experiments for WT and H1085Y Pol II are
shown in the left and right panels, respectively. Average
elongation rates for each NTP concentration were measured as the
length of the transcribed region (51 nt) divided by the time of
half-maximal accumulation of run-off product (61 nt). Average
elongation rates were then plotted versus NTP concentration to
infer maximum average elongation rate (see Experimental
Procedures for details) (top right graph). Inferred maximum
average elongation rates are shown in the bottom right graph
with error bars representing the 95% confidence interval (See
Experimental Procedures for details).
(B) H1085Y Pol II
exhibits only modest defects for 2′-dNTP incorporation. WT and
H1085Y Pol II ECs were formed on oligonucleotide scaffolds
containing 10-mer RNAs with templates specifying addition of
different NTPs at position 11. Average incorporation rates for
different template-specified 2′-dNTPs were measured as
1/t[1/2] for maximal accumulation of 11-mer RNA. Incorporation
rates were then plotted versus 2′-dNTP concentration, and
maximum incorporation rate for either 2′-dATP or 2′-dGTP was
inferred (left panels). Maximum incorporation rates for WT Pol
II and H1085Y are shown in the right panels with error bars
representing the 95% confidence interval (See Experimental
Procedures for details).
(C) H1085Y Pol II exhibits modest
defects in GTP misincorporation. WT and H1085Y ECs were formed
and labeled as in (B) with templates specifying incorporation of
ATP at the position being measured, but were challenged with 1
mM GTP and misincorporation rate measured as the 1/t[1/2] for
maximal incorporation. Mean misincorporation rate from at least
three experiments is represented in the bar graph (error bars
represent ± SD).
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Figure 7.
Figure 7. Direct Interaction between Rpb1 His1085 and
α-Amanitin and TL Capture May Underlie α-Amanitin Inhibition
of Transcription
(A) Overall view of α-amanitin and the
new TL conformation and their positions in relation to the
Bridge helix (BH). A superpositioned EC structure (PDB 2E2H)
showing DNA (magenta), RNA (red), nontemplate DNA (green), and
nucleotide GTP (orange) highlights the position of the inhibitor
and TL in relation to EC components.
(B) A 90° rotation
shows the α-amanitin position in relation to the Bridge helix
(BH) and its capture of the TL Rpb1 His1085.
(C) TL
residues Rpb1 1084–1086 and the entire α-amanitin modeled
into electron density (dark gray mesh) from an initial unbiased
2Fo-Fc electron density map contoured at 0.6 σ.
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The above figures are
reprinted
from an Open Access publication published by Cell Press:
Mol Cell
(2008,
30,
547-556)
copyright 2008.
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