Inhibitor Tagetitoxin in Complex with T.Thermophilus RNA Polymerase

Faiza Sulaiman '15 and Kalkidan Aseged '17


Contents:


I. Introduction

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 through photosynthesis.

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 Alpha, Alpha (which come together to form an Alpha Dimer), Beta, Beta', 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


Tagetitoxin (C11H17N2 O11 P S) is a bicyclic compound with two-fused six-membered heterocyclic rings. 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 and tMG 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, Tgtís 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.

E 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.

Pre-Insertion 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 pre-insertion site.

Insertion Site
Similar to its interaction with the pre-insertion site, Tgt does not 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 of RNAP

VI. Implications



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.


VI. References

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