Tagetitoxin inhibits RNA polymerase through trapping of the trigger loop

Abstract

Tagetitoxin (Tgt) inhibits multisubunit chloroplast, bacterial, and some eukaryotic RNA polymerases (RNAPs). A crystallographic structure of Tgt bound to bacterial RNAP apoenzyme shows that Tgt binds near the active site but does not explain why Tgt acts only at certain sites. To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex. In this model, Tgt interacts with the β' subunit trigger loop (TL), stabilizing it in an inactive conformation. We show that (i) substitutions of the Arg residue of TL contacted by Tgt confer resistance to inhibitor; (ii) Tgt inhibits RNAP translocation, which requires TL movements; and (iii) paused complexes and a "slow" enzyme, in which the TL likely folds into an altered conformation, are resistant to Tgt. Our studies highlight the role of TL as a target through which accessory proteins and antibiotics can alter the elongation complex dynamics.

FIGURE 1.

Tgt in a co-crystal with Tth RNAP holoenzyme (Protein Data Bank code 2BE5). Tgt is shown as purple sticks, the β′ BH and TL as teal and orange schemes, respectively. The catalytic Mg²⁺ ion is indicated by a red sphere, the catalytic Asp triad by dark blue sticks.

FIGURE 2.

Paused transcription complexes are resistant to Tgt. Radiolabeled halted G37 and A29 complexes were formed on pIA171 (A) and pIA349 (B) templates. These template encode the his and the ops pause signals (shown below). Halted were incubated with Tgt (5 μm) for 2 min at 37 °C and challenged with NTPs and heparin. Aliquots were withdrawn at the indicated times and separated on a 10% denaturing gel. Positions of the halted RNAs, the paused RNA species (U43 at ops, U71 at his site), the run-off transcripts and several other RNA products are indicated with arrows.

FIGURE 3.

Fast and slow enzymes respond differently to Tgt. Top, radiolabeled A29 complexes formed on pIA171 template in the presence of increased concentrations of Tgt (indicated above each lane) with the wild-type (left), RpoB5101 (center), or RpoB8 (right) RNAPs during a 15-min incubation at 37 °C. Reactions were quenched and analyzed on a 10% denaturing gel. Bottom, Distances (in Å) between crystallographic (Protein Data Bank code 2BE5) Tgt (shown as purple sticks and semitransparent spheres) binding site and the positions of fast (blue) and slow (red) amino acid substitutions in RNAP. The residues are numbered as in E. coli.

FIGURE 4.

Tgt interacts with the TL in the Tth EC model. RNA (red scheme), nontemplate DNA (blue scheme), template DNA (black scheme), and substrate NTP (green sticks) are shown; the other colors are as in Fig. 1.

FIGURE 5.

Substitutions of Eco β′Arg-933 (Tth Arg-1239) abolish inhibition by Tgt. A, polar contacts between Tgt (purple sticks) and amino acids (orange and gray sticks) in the model of Tgt/EC (shown in Fig. 4; the color scheme is preserved). B and C, inhibition of abortive transcription on the T7A1 promoter by Tgt. Formation of the radiolabeled ApUpC RNA was followed as a function of Tgt concentration (from 0 to 32 μm) with the wild-type or altered Eco RNAPs. The key is shown in the figure.

FIGURE 6.

Tgt inhibits RNAP translocation. The translocation registers of the EC35 formed on a template shown on top with the wild-type or β′R933A Eco RNAPs determined by ExoIII footprinting; Tgt (60 μm) and AMP (1 mm) were added where indicated. The stop positions that correspond to the post- and pretranslocated ECs are shown by arrows, and their ratios are shown below each lane. See also supplemental Fig. S4.