Inhibitor Tagetitoxin in Complex with
T.Thermophilus RNA Polymerase
Faiza Sulaiman '15 and Kalkidan Aseged '17
Tagetitoxin (Tgt) is a bacterial phytotoxin produced by a
common plant pathogen named Pseudomonas syringae pv. tagetis.
In vitro, the production of Tgt leads to the inhibition of bacterial
RNA Polymerase (RNAP) and RNAP III in eukaryotes. In vivo, TGT
naturally targets and inhibits chloroplast RNAP in plants. The
inhibition of chloroplast RNAP results in apical
chlorosis, which ultimately prevents chloroplast formation. In
the absence of chloroplasts, plants lose their green color and more
importantly, are unable to produce carbohydrates and other nutrients
The specific mechanism through which Tgt inhibits RNAP is
not completely clear. However, analysis of its structure and manner
of binding to RNAP suggests that Tgt inhibits RNAP not through
direct competition with NTPs, but by stabilizing the NTP substrate
in an inactive conformation. Furthermore, interactions with
magnesium ions near the RNAP active site (cMG2 and tMG) assist Tgt
in its inhibition of RNAP. Below, we will explore the manner in
which TGT may bind and inhibit the T. thermophilus
RNAP in more detail.
II. RNA Polymerase Structure and Function
RNA Polymerase is the enzyme which catalyzes transcription,
or the synthesis of RNA from a DNA template. Transcription is the
first major step of gene expression, which is the process by which
genes code for functional products such as proteins. The core enzyme
of RNAP consists of
(which come together to form an Alpha Dimer),
and Omega. It is only when the
initiation factor sigma binds to the core enzyme as a 6th
that the RNAP holenzyme forms. This holoenzyme is the active
form of RNAP required to initiate catalysis of transcription. Once
the holoenzyme is formed, it binds to the promotor
region of double stranded DNA while in a "closed complex."
Once bound, RNAP undergoes isomerization, which is the
transition of the enzyme from the the closed complex to the open
complex. During this transition, a region in the sigma subunit
changes conformation so that it is no longer blocking the
, labeled here by cMG1, one of
two catalytic Mg2+ ions of RNAP. With the active site open, the
template DNA can then be fed through and base paired with incoming
NTPs so that it can be transcribed into RNA. Various channels in the
enzyme allow for the movement of the template DNA and the
transcribed RNA during catalysis. These channels
include the NTP entry channel, RNA Exit Channel, DNA Entry
Channel, and the DNA Exit Channel.
III. Tagetitoxin Structure
O11 P S)
is a bicyclic compound with two-fused six-membered heterocyclic
The RNAP inhibitor Tgt contains many electrophillic atoms (oxygen,
nitrogen, phosphorous, and sulfur) which allow it to participate
in polar interactions with neighboring residues and catalytic
Mg2+ atoms in the RNAP active site.
IV. Tgt-RNAP Complex
When complexed with the RNA Polymerase of T.
thermophilus, Tgt binds at the base of the secondary
(NTP entry) channel,
to RNAP's active site (cMG1).
Binding between Tgt and RNAP holoenzyme is
facilitated soley through polar interactions. Nine out of
the eleven oxygens in Tgt interact with
, forming a total of 18 hydrogen bonds. However, the most
important of these hydrogen bonds occur with the
B Arg678, B
Arg1106, B' Arg731,
and B' Asn458, the last of which
assists with NTP recognition.
In addition to the hydrogen bonds formed with adjacent
protein side chains, the fixed Mg2+ ion in RNAP, tMG,
is used to stabalize the Tgt-RNAP complex. tMG is
coordinated by a TGT phosphate and two active site residues,
B' Asp460 and B
Glu813. These residues also help bridge cMG1
to be better stabilized in the complex.
Interestingly, while Tgt actually increases RNAP
affinity for cMG1, the major catalytic Mg2+ ion, tMG impairs
binding of cMG2, the second Mg2+ ion needed for catalysis of
transcription. The inability of cMG2 (not shown) to bind to
the catalytic site decreases the rate of transcription by
impairing NTP stabilization.
V. Mechanism of Tgt Inhibition
Rather than directly competing with incoming NTPs,
Tgt’s placement in RNAP suggests that it influences the
structure of an NTP by stabilizing it into an inactive
state in one of the three sites for substrate loading:
Entry (E) Site, Pre-Insertion Site, and Insertion Site.
The E-site overlaps
significantly with the Tgt binding site, suggesting
that Tgt could have an influence on NTP loading in this site.
However, the interaction between NTPs and the E site is very
weak because it is a non-specific inactive site. So, it is
unlikely that Tgt would be able to inhibit transcription if it
only acted upon this site.
In the pre-insertion site, NTPs are recognized through base
pairing with the template DNA strand. Tgt and the
pre-insertion site have a slight
steric clash, however rotations of the beta and gamma
phoshphate groups eliminate any clash, arguing against the
idea that Tgt inhibits RNAP through interaction with the
Similar to its interaction with the pre-insertion site, Tgt
not significantly overlap with the insertion site.
However, in this site, the Beta' Asp460 amino acid is
exchanged for tMG, which then can compete for interaction with
the Tgt gamma phosphate. This competition would result in NTP
base pairing with the DNA template, but its phosphates would
remain in an inactive form. tMG interacting with the gamma
phosphate would also increase cMG1 affinity for this inactive
form of the NTP. Therefore, it is proposed that interaction
between Tgt and the insertion site is what leads to the inhibition
There are a wide variety of other factors known to
alter the activity of the RNAP active site through the
secondary (NTP entry) channel. TGT's binding at the base of
this channel confirms the channel's role as an important
target for transcriptional regulation. Another RNAP inhibitor
which makes use this of channel is ppGpp, an alarmone involved
in repressing RNAP in bacteria such as E. coli.
Modeling of ppGpp shows that its binding site not only overlaps
with the Tgt binding site, but also the two repressors are
very similar in structure, with only a few differences. The overlap between the two binding sites
suggests that Tgt and ppGpp compete for binding to RNAP, and
this has been supported by past experiments.
Additionally, other studies have suggested that
misalignment of Mg2+ ions of the active site is a very
common method of altering catalysis of nucleic acid enzymes.
For example, similar to TGT's use of tMG, RNaseH inhibitors
are known to recruit additional Mg2+ ions to disrupt
coordination of the catalytic Mg2+ ions.
These are very interesting observations as they imply that
the structural development of many different types of
nucleic acid inhibitors may have been driven by convergent
evolution. That is, inhibitor designs may have been molded
by similar evolutionary pressures to inhibit similar nucleic
acid enzymes. Importantly, strengthening understanding of
the inhibitory mechanism of TGT and other naturally occuring
inhibitors of RNAP could provide opportunities for
developing new artificial/ drug designs to regulate
transcription. Insight on the mechanism of inhibition may
also help design inhibitors for an assortment of other
similar nucleic acid enzymes.
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