pyrophosphate and tagetitoxin

Transcription elongation consists of repetition of the nucleotide addition cycle: phosphodiester bond formation, translocation and binding of the next nucleotide. Inhibitor of multi-subunit RNA polymerase tagetitoxin (TGT) enigmatically slows down addition of nucleotides in a sequence-dependent manner, only at certain positions of the template. Here, we show that TGT neither affects chemistry of RNA synthesis nor induces backward translocation, nor competes with the nucleoside triphosphate (NTP) in the active center. Instead, TGT increases the stability of the pre-translocated state of elongation complex, thus slowing down addition of the following nucleotide. We show that the extent of inhibition directly depends on the intrinsic stability of the pre-translocated state. The dependence of translocation equilibrium on the transcribed sequence results in a wide distribution (~1-10(3)-fold) of inhibitory effects of TGT at different positions of the template, thus explaining sequence-specificity of TGT action. We provide biochemical evidence that, in pre-translocated state, TGT stabilizes folded conformation of the Trigger Loop, which inhibits forward and backward translocation of the complex. The results suggest that Trigger Loop folding in the pre-translocated state may serve to reduce back-tracking of the elongation complex. Overall, we propose that translocation may be a limiting and highly regulated step of RNA synthesis.

Topics: Dicarboxylic Acids; Diphosphates; DNA-Directed RNA Polymerases; Nucleic Acid Synthesis Inhibitors; Organophosphorus Compounds; Transcription Elongation, Genetic; Transcriptional Elongation Factors

Multisubunit RNA polymerase (RNAP) is the central information-processing enzyme in all cellular life forms, yet its mechanism of translocation along the DNA molecule remains conjectural. Here, we report direct monitoring of bacterial RNAP translocation following the addition of a single nucleotide. Time-resolved measurements demonstrated that translocation is delayed relative to nucleotide incorporation and occurs shortly after or concurrently with pyrophosphate release. An investigation of translocation equilibrium suggested that the strength of interactions between RNA 3' nucleotide and nucleophilic and substrate sites determines the translocation state of transcription elongation complexes, whereas active site opening and closure modulate the affinity of the substrate site, thereby favoring the post- and pre-translocated states, respectively. The RNAP translocation mechanism is exploited by the antibiotic tagetitoxin, which mimics pyrophosphate and induces backward translocation by closing the active site.

Topics: Bacteria; Catalytic Domain; Dicarboxylic Acids; Diphosphates; DNA; DNA-Directed RNA Polymerases; Enzyme Inhibitors; Fluorescent Dyes; Nucleotides; Organophosphorus Compounds; Protein Subunits; Protein Transport; RNA; Transcription, Genetic