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.

Membrane-bound pyrophosphatases (mPPases), which couple pyrophosphate hydrolysis to transmembrane transport of H and/or Na ions, are divided into K,Na-independent, Na-regulated, and K-dependent families. The first two families include H-transporting mPPases (H-PPases), whereas the last family comprises one Na-transporting, two Na- and H-transporting subfamilies (Na-PPases and Na,H-PPases, respectively), and three H-transporting subfamilies. Earlier studies of the few available model mPPases suggested that K binds to a site located adjacent to the pyrophosphate-binding site, but is substituted by the ε-amino group of an evolutionarily acquired lysine residue in the K-independent mPPases. Here, we performed a systematic analysis of the K/Lys cationic center across all mPPase subfamilies. An Ala → Lys replacement in K-dependent mPPases abolished the K dependence of hydrolysis and transport activities and decreased these activities close to the level (4-7%) observed for wild-type enzymes in the absence of monovalent cations. In contrast, a Lys → Ala replacement in K,Na-independent mPPases conferred partial K dependence on the enzyme by unmasking an otherwise conserved K-binding site. Na could partially replace K as an activator of K-dependent mPPases and the Lys → Ala variants of K,Na-independent mPPases. Finally, we found that all mPPases were inhibited by excess substrate, suggesting strong negative co-operativity of active site functioning in these homodimeric enzymes; moreover, the K/Lys center was identified as part of the mechanism underlying this effect. These findings suggest that the mPPase homodimer possesses an asymmetry of active site performance that may be an ancient prototype of the rotational binding-change mechanism of F-type ATPases.

One of the strategies used by organisms to adapt to life under conditions of short energy supply is to use the by-product pyrophosphate to support cation gradients in membranes. Transport reactions are catalyzed by membrane-integral pyrophosphatases (PPases), which are classified into two homologous subfamilies: H + -transporting (found in prokaryotes, protists, and plants) and Na + -transporting (found in prokaryotes). Transport activities have been believed to require specific machinery for each ion, in accordance with the prevailing paradigm in membrane transport. However, experiments using a fluorescent pH probe and 22 Na + measurements in the current study revealed that five bacterial PPases expressed in Escherichia coli have the ability to simultaneously translocate H + and Na + into inverted membrane vesicles under physiological conditions. Consistent with data from phylogenetic analyses, our results support the existence of a third, dual-specificity bacterial Na + ,H + -PPase subfamily, which apparently evolved from Na + -PPases. Interestingly, genes for Na + ,H + -PPase have been found in the major microbes colonizing the human gastrointestinal tract. The Na + ,H + -PPases require Na + for hydrolytic and transport activities and are further activated by K + . Based on ionophore effects, we conclude that the Na + and H + transport reactions are electrogenic and do not result from secondary antiport effects. Sequence comparisons further disclosed four Na + ,H + -PPase signature residues located outside the ion conductance channel identified earlier in PPases using X-ray crystallography. Our results collectively support the emerging paradigm that both Na + and H + can be transported via the same mechanism, with switching between Na + and H + specificities requiring only subtle changes in the transporter structure.

Bacterial RNA polymerase (RNAP) is a validated target for antibacterial drugs. CBR703 series antimicrobials allosterically inhibit transcription by binding to a conserved α helix (β′ bridge helix, BH) that interconnects the two largest RNAP subunits. Here we show that disruption of the BH-β subunit contacts by amino-acid substitutions invariably results in accelerated catalysis, slowed-down forward translocation and insensitivity to regulatory pauses. CBR703 partially reverses these effects in CBR-resistant RNAPs while inhibiting catalysis and promoting pausing in CBR-sensitive RNAPs. The differential response of variant RNAPs to CBR703 suggests that the inhibitor binds in a cavity walled by the BH, the β′ F-loop and the β fork loop. Collectively, our data are consistent with a model in which the β subunit fine tunes RNAP elongation activities by altering the BH conformation, whereas CBRs deregulate transcription by increasing coupling between the BH and the β subunit.

Membrane-bound pyrophosphatase (PPase) is commonly believed to couple pyrophosphate (PPi) hydrolysis to H+ transport across the membrane. Here, we demonstrate that two newly isolated bacterial membrane PPases from the mesophile Methanosarcina mazei (Mm-PPase) and the moderate thermophile Moorella thermoacetica and a previously described PPase from the hyperthermophilic bacterium Thermotoga maritima catalyze Na+ rather than H+ transport into Escherichia coli inner membrane vesicles (IMV). When assayed in uncoupled IMV, the three PPases exhibit an absolute requirement for Na+ but display the highest hydrolyzing activity in the presence of both Na+ and K+. Steady-state kinetic analysis of PPi hydrolysis by Mm-PPase revealed two Na+ binding sites. One of these sites can also bind K+, resulting in a 10-fold increase in the affinity of the other site for Na+ and a 2-fold increase in maximal velocity. PPi-driven 22Na+ transport into IMV containing Mm-PPase was unaffected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, inhibited by the Na+ ionophore monensin, and activated by the K+ ionophore valinomycin. The Na+ transport was accompanied by the generation of a positive inside membrane potential as reported by Oxonol VI. These findings define Na+-dependent PPases as electrogenic Na+ pumps. Phylogenetic analysis suggests that ancient gene duplication preceded the split of Na+- and H+-PPases.

SUMMARY In its early history, life appeared to depend on pyrophosphate rather than ATP as the source of energy. Ancient membrane pyrophosphatases that couple pyrophosphate hydrolysis to active H + transport across biological membranes (H + -pyrophosphatases) have long been known in prokaryotes, plants, and protists. Recent studies have identified two evolutionarily related and widespread prokaryotic relics that can pump Na + (Na + -pyrophosphatase) or both Na + and H + (Na + ,H + -pyrophosphatase). Both these transporters require Na + for pyrophosphate hydrolysis and are further activated by K + . The determination of the three-dimensional structures of H + - and Na + -pyrophosphatases has been another recent breakthrough in the studies of these cation pumps. Structural and functional studies have highlighted the major determinants of the cation specificities of membrane pyrophosphatases and their potential use in constructing transgenic stress-resistant organisms.