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Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor $\sigma$ to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein-DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RNAP-promoter open (RPO) complex is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with $\sigma$ and the RNAP $\beta$ subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

Here we describe a direct fluorescence method that reports real-time occupancies of the pre- and post-translocated state of multisubunit RNA polymerase. In a stopped-flow setup, this method is capable of resolving a single base-pair translocation motion of RNA polymerase in real time. In a conventional spectrofluorometer, this method can be employed for studies of the time-averaged distribution of RNA polymerase on the DNA template. This method utilizes commercially available base analogue fluorophores integrated into template DNA strand in place of natural bases. We describe two template DNA strand designs where translocation of RNA polymerase from a pre-translocation to a post-translocation state results in disruption of stacking interactions of fluorophore with neighboring bases, with a concomitant large increase in fluorescence intensity.

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. [ABSTRACT FROM AUTHOR]

Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thennotoga maritima (Tm-PPase), a homologue of H+ pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H+-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na+ but displayed the highest activity in the presence of millimolar levels of both Na+ and K+. Detergent-solubilized recombinant Tm-PPase was thermo stable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady- state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na+ binding sites and one K+ binding site are involved in enzyme activation. The affinity of the site that binds Na+ first is increased with increasing K+ concentration. In contrast, only one Na+ binding site (K+-dependent) and one K+ binding site were involved in activation of the Asp703 → Asn variant. Thus, Asp703 may form part of the K+-independent Na+ binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PPi-synthesizing activity, which also required Na+ but was inhibited by K+ These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na+edependent H+-PPase homologues and may be an analogue of NatK+-ATPase. [ABSTRACT FROM AUTHOR]

H+-pyrophosphatase (H+-PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H+-PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu197, Glu202, Glu550, and Glu649 in the H+-PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PPi hydrolysis and PPi-energized proton translocation, indicating that the negative charge of Glu202 is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg2+ concentrations but were only slightly affected at high Mg2+ concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two- and three-metal pathways. Substitution of Glu197, Glu202, and Glu649 resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methyl- enediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu550 and Glu649, R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PPi hydrolysis to proton transport. [ABSTRACT FROM AUTHOR]

Membrane pyrophosphatases (PPases), divided into K+-dependent and K+-independent subfamilies, were believed to pump H+ across cell membranes until a recent demonstration that some K+-dependent PPases function as Na+ pumps. Here, we have expressed seven evolutionarily important putative PPases in Escherichia coli and estimated their hydrolytic, Na+ transport, and H+ transport activities as well as their K+ and Na+ requirements in inner membrane vesicles. Four of these enzymes (from Anaerostipes caccae, Chlorobium limicola, Clostridium tetani, and Desulfuromonas acetoxidans) were identified as K+-dependent Na+ transporters. Phylogenetic analysis led to the identification of a monophyletic clade comprising characterized and predicted Na+-transporting PPases (Na+-PPases) within the K+-dependent subfamily. H+-transporting PPases (H+-PPases) are more heterogeneous and form at least three independent clades in both subfamilies. These results suggest that rather than being a curious rarity, Na+-PPases predominantly constitute the K+-dependent subfamily. Furthermore, Na+-PPases possibly preceded H+-PPases in evolution, and transition from Na+ to H+ transport may have occurred in 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. [ABSTRACT FROM AUTHOR]