Anssi M. Malinen

Active site opening and closure control translocation of multisubunit RNA polymerase

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.

Na+-translocating Membrane Pyrophosphatases Are Widespread in the Microbial World and Evolutionarily Precede H+-translocating Pyrophosphatases

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.

Membrane-integral pyrophosphatase subfamily capable of translocating both Na+ and H+

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 22Na+ 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.

Pyrophosphate-Fueled Na+ and H+ Transport in Prokaryotes

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.

Membrane Na+-pyrophosphatases can transport protons at low sodium concentrations

Background: Prokaryotic membrane Na+-PPases couple PPi hydrolysis with active Na+ transport. Results: Na+-PPases can transport both Na+ and H+ at Na+ concentrations <5 mm but only Na+ above this level. Conclusion: Na+-PPases are dual specificity transporters in which H+ transport activity is masked under physiological conditions. Significance: Inherent Na+/H+ promiscuity provides a basis for Na+-PPase evolution to H+-PPase. Membrane-bound Na+-pyrophosphatase (Na+-PPase), working in parallel with the corresponding ATP-energized pumps, catalyzes active Na+ transport in bacteria and archaea. Each ∼75-kDa subunit of homodimeric Na+-PPase forms an unusual funnel-like structure with a catalytic site in the cytoplasmic part and a hydrophilic gated channel in the membrane. Here, we show that at subphysiological Na+ concentrations (<5 mm), the Na+-PPases of Chlorobium limicola, four other bacteria, and one archaeon additionally exhibit an H+-pumping activity in inverted membrane vesicles prepared from recombinant Escherichia coli strains. H+ accumulation in vesicles was measured with fluorescent pH indicators. At pH 6.2–8.2, H+ transport activity was high at 0.1 mm Na+ but decreased progressively with increasing Na+ concentrations until virtually disappearing at 5 mm Na+. In contrast, 22Na+ transport activity changed little over a Na+ concentration range of 0.05–10 mm. Conservative substitutions of gate Glu242 and nearby Ser243 and Asn677 residues reduced the catalytic and transport functions of the enzyme but did not affect the Na+ dependence of H+ transport, whereas a Lys681 substitution abolished H+ (but not Na+) transport. All four substitutions markedly decreased PPase affinity for the activating Na+ ion. These results are interpreted in terms of a model that assumes the presence of two Na+-binding sites in the channel: one associated with the gate and controlling all enzyme activities and the other located at a distance and controlling only H+ transport activity. The inherent H+ transport activity of Na+-PPase provides a rationale for its easy evolution toward specific H+ transport.

Mutual effects of cationic ligands and substrate on activity of the Na+-transporting pyrophosphatase of Methanosarcina mazei.

The PP(i)-driven sodium pump (membrane pyrophosphatase) of Methanosarcina mazei (Mm-PPase) absolutely requires Na(+) and Mg(2+) for activity and additionally employs K(+) as a modulating cation. Here we explore relationships among Na(+), K(+), Mg(2+), and PP(i) binding sites by analyzing the dependency of the Mm-PPase PP(i)-hydrolyzing function on these ligands and protection offered by the ligands against Mm-PPase inactivation by trypsin and the SH-reagent mersalyl. Steady-state kinetic analysis of PP(i) hydrolysis indicated that catalysis involves random order binding of two Mg(2+) ions and two Na(+) ions, and the binding was almost independent of substrate (Mg(2)PP(i) complex) attachment. Each pair of metal ions, however, binds in a positively cooperative (or ordered) manner. The apparent cooperativity is lost only when Na(+) binds to preformed enzyme-Mg(2+)-substrate complex. The binding of K(+) increases, by a factor of 2.5, the catalytic constant, the Michaelis constant, and the Mg(2+) binding affinity, and these effects may result from K(+) binding to either one of the Na(+) sites or to a separate site. The effects of ligands on Mm-PPase inactivation by mersalyl and trypsin are highly correlated and are strongly indicative of ligand-induced enzyme conformational changes. Importantly, Na(+) binding induces a conformational change only when completing formation of the catalytically competent enzyme-substrate complex or a similar complex with a diphosphonate substrate analog. These data indicate considerable flexibility in Mm-PPase structure and provide evidence for its cyclic change in the course of catalytic turnover.

CBR antimicrobials alter coupling between the bridge helix and the β subunit in RNA polymerase

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.

Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase

Significance The majority of aerobic living organisms use oxygen for respiration. The key enzyme, which directly reduces oxygen to water during respiration, is the terminal cytochrome c oxidase. It generates a large portion of the utilizable energy provided by the respiratory chain. Accumulation of biologically available energy by means of cytochrome c oxidases is believed to be due to the proton-motive force across the mitochondrial or bacterial membrane. Details of this energy conversion are still unclear. Here we report the discovery of a sodium-pumping cytochrome c oxidase that converts energy of respiration into sodium-motive force. This finding provides clues to understanding the mechanism of cytochrome c oxidase that is not available when applying knowledge of the proton-pumping versions of the enzyme. Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which are widespread among pathogenic bacteria, are the least understood. In particular, the proton-pumping machinery of these Coxs has not yet been elucidated despite the availability of X-ray structure information. Here, we report the discovery of the first (to our knowledge) sodium-pumping Cox (Scox), a cbb3 cytochrome from the extremely alkaliphilic bacterium Thioalkalivibrio versutus. This finding offers clues to the previously unknown structure of the ion-pumping channel in the C-type Coxs and provides insight into the functional properties of this enzyme.

Monitoring Translocation of Multisubunit RNA Polymerase Along the DNA with Fluorescent Base Analogues

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.

Two independent evolutionary routes to Na+/H+ co-transport function in membrane pyrophosphatases

Membrane-bound pyrophosphatases (mPPases) hydrolyze pyrophosphate (PPi) to transport H(+), Na(+) or both and help organisms to cope with stress conditions, such as high salinity or limiting nutrients. Recent elucidation of mPPase structure and identification of subfamilies that have fully or partially switched from Na(+) to H(+) pumping have established mPPases as versatile models for studying the principles governing the mechanism, specificity and evolution of cation transporters. In the present study, we constructed an accurate phylogenetic map of the interface of Na(+)-transporting PPases (Na(+)-PPases) and Na(+)- and H(+)-transporting PPases (Na(+),H(+)-PPases), which guided our experimental exploration of the variations in PPi hydrolysis and ion transport activities during evolution. Surprisingly, we identified two mPPase lineages that independently acquired physiologically significant Na(+) and H(+) cotransport function. Na(+),H(+)-PPases of the first lineage transport H(+) over an extended [Na(+)] range, but progressively lose H(+) transport efficiency at high [Na(+)]. In contrast, H(+)-transport by Na(+),H(+)-PPases of the second lineage is not inhibited by up to 100 mM Na(+) With the identification of Na(+),H(+)-PPase subtypes, the mPPases protein superfamily appears as a continuum, ranging from monospecific Na(+) transporters to transporters with tunable levels of Na(+) and H(+) cotransport and further to monospecific H(+) transporters. Our results lend credence to the concept that Na(+) and H(+) are transported by similar mechanisms, allowing the relative efficiencies of Na(+) and H(+) transport to be modulated by minor changes in protein structure during the course of adaptation to a changing environment.