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.

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.

several independent enzyme lineages. Site-directed mutagenesis studies facilitated the identification of a specific Glu residue that appears to be central in the transport mechanism. This residue is located in the cytoplasm-membrane interface of transmembrane helix 6 in Na ؉ -PPases but shifted to within the membrane or helix 5 in H ؉ -PPases. These results contribute to the prediction of the transport specificity and K ؉ dependence for a particular membrane PPase sequence based on its position in the phylogenetic tree, identity of residues in the K ؉ dependence signature, and position of the membrane-located Glu residue.