Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domain.

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a ß-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length and is dispensable for DNA supercoiling, the ß-bulge loop is required for supercoiling activity. It has previously been shown that the ß-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a ß-bulge loop as a minimal latch is reported. It is shown that the ß-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.

The domains of yeast eIF4G, eIF4E and the cap fine-tune eIF4A activities through an intricate network of stimulatory and inhibitory effects.

Translation initiation in eukaryotes starts with the recognition of the mRNA 5'-cap by eIF4F, a hetero-trimeric complex of eIF4E, the cap-binding protein, eIF4A, a DEAD-box helicase, and eIF4G, a scaffold protein. eIF4G comprises eIF4E- and eIF4A-binding domains (4E-BD, 4A-BD) and three RNA-binding regions (RNA1-RNA3), and interacts with eIF4A, eIF4E, and with the mRNA. Within the eIF4F complex, the helicase activity of eIF4A is increased. We showed previously that RNA3 of eIF4G is important for the stimulation of the eIF4A conformational cycle and its ATPase and helicase activities. Here, we dissect the interplay between the eIF4G domains and the role of the eIF4E/cap interaction in eIF4A activation. We show that RNA2 leads to an increase in the fraction of eIF4A in the closed state, an increased RNA affinity, and faster RNA unwinding. This stimulatory effect is partially reduced when the 4E-BD is present. eIF4E binding to the 4E-BD then further inhibits the helicase activity and closing of eIF4A, but does not affect the RNA-stimulated ATPase activity of eIF4A. The 5'-cap renders the functional interaction of mRNA with eIF4A less efficient. Overall, the activity of eIF4A at the 5'-cap is thus fine-tuned by a delicately balanced network of stimulatory and inhibitory interactions.

Conjugates of Ciprofloxacin and Amphiphilic Block Copoly(2-alkyl-2-oxazolines)s Overcome Efflux Pumps and Are Active against CIP-Resistant Bacteria.

Conjugation of antibiotics with polymers is an emerging strategy to improve the performance of these important drugs. Here, the antibiotic ciprofloxacin (CIP) was conjugated with amphiphilic poly(2-oxazoline) (POx) block copolymers to investigate whether the activity of the antibiotic was enhanced due to additionally induced membrane activity. The resulting polymer-antibiotic conjugates (PACs) are an order of magnitude more active against the bacterial strain Staphylococcus aureus than CIP and show high activities against numerous pathogenic bacterial strains. Their high activity depends on an optimal hydrophobic/hydrophilic balance (HHB) of the POx tail. Mechanistic studies revealed that the derivatization of CIP required for the polymer conjugation lowers the affinity of the antibiotic to its target topoisomerase IV. However, the amphiphilic PACs are most likely concentrated within the bacterial cytoplasm, which overcompensates the loss of affinity and results in high antibacterial activity. In addition, the development of resistance in S. aureus and Escherichia coli is slowed down. More importantly, the amphiphilic PACs are active against CIP-resistant S. aureus and E. coli. The PACs with the highest activity are not cytotoxic toward human stem cells and do not lyse blood cells in saturated solution.

A ß-hairpin is a Minimal Latch that Supports Positive Supercoiling by Reverse Gyrase.

Reverse gyrase is a unique type I topoisomerase that catalyzes the introduction of positive supercoils into DNA in an ATP-dependent reaction. Supercoiling is the result of a functional cooperation of the N-terminal helicase domain with the C-terminal topoisomerase domain. The helicase domain is a nucleotide-dependent conformational switch that alternates between open and closed states with different affinities for single- and double-stranded DNA. The isolated helicase domain as well as full-length reverse gyrase can transiently unwind double-stranded regions in an ATP-dependent reaction. The latch region of reverse gyrase, an insertion into the helicase domain with little conservation in sequence and length, has been proposed to coordinate events in the helicase domain with strand passage by the topoisomerase domain. Latch deletions lead to a reduction in or complete loss of supercoiling activity. Here we show that the latch consists of two functional parts, a globular domain that is dispensable for DNA supercoiling and a ß-hairpin that connects the globular domain to the helicase domain and is required for supercoiling activity. The ß-hairpin thus constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain.

Functional interactions between gyrase subunits are optimized in a species-specific manner.

DNA gyrase is a bacterial DNA topoisomerase that catalyzes ATP-dependent negative DNA supercoiling and DNA decatenation. The enzyme is a heterotetramer comprising two GyrA and two GyrB subunits. Its overall architecture is conserved, but species-specific elements in the two subunits are thought to optimize subunit interaction and enzyme function. Toward understanding the roles of these different elements, we compared the activities of Bacillus subtilis, Escherichia coli, and Mycobacterium tuberculosis gyrases and of heterologous enzymes reconstituted from subunits of two different species. We show that B. subtilis and E. coli gyrases are proficient DNA-stimulated ATPases and efficiently supercoil and decatenate DNA. In contrast, M. tuberculosis gyrase hydrolyzes ATP only slowly and is a poor supercoiling enzyme and decatenase. The heterologous enzymes are generally less active than their homologous counterparts. The only exception is a gyrase reconstituted from mycobacterial GyrA and B. subtilis GyrB, which exceeds the activity of M. tuberculosis gyrase and reaches the activity of the B. subtilis gyrase, indicating that the activities of enzymes containing mycobacterial GyrB are limited by ATP hydrolysis. The activity pattern of heterologous gyrases is in agreement with structural features present: B. subtilis gyrase is a minimal enzyme, and its subunits can functionally interact with subunits from other bacteria. In contrast, the specific insertions in E. coli and mycobacterial gyrase subunits appear to prevent efficient functional interactions with heterologous subunits. Understanding the molecular details of gyrase adaptations to the specific physiological requirements of the respective organism might aid in the development of species-specific gyrase inhibitors.

Single-stranded regions modulate conformational dynamics and ATPase activity of eIF4A to optimize 5'-UTR unwinding.

Eukaryotic translation initiation requires unwinding of secondary structures in the 5'-untranslated region of mRNA. The DEAD-box helicase eIF4A is thought to unwind structural elements in the 5'-UTR in conjunction with eIF4G and eIF4B. Both factors jointly stimulate eIF4A activities by modulation of eIF4A conformational cycling between open and closed states. Here we examine how RNA substrates modulate eIF4A activities. The RNAs fall into two classes: Short RNAs only partially stimulate the eIF4A ATPase activity, and closing is rate-limiting for the conformational cycle. By contrast, longer RNAs maximally stimulate ATP hydrolysis and promote closing of eIF4A. Strikingly, the rate constants of unwinding do not correlate with the length of a single-stranded region preceding a duplex, but reach a maximum for RNA with a single-stranded region of six nucleotides. We propose a model in which RNA substrates affect eIF4A activities by modulating the kinetic partitioning of eIF4A between futile, unproductive, and productive cycles.

Gyrase containing a single C-terminal domain catalyzes negative supercoiling of DNA by decreasing the linking number in steps of two.

The topological state of DNA in vivo is regulated by topoisomerases. Gyrase is a bacterial topoisomerase that introduces negative supercoils into DNA at the expense of ATP hydrolysis. According to the strand-passage mechanism, a double-strand of the DNA substrate is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. The correct orientation of these DNA segments for strand passage is achieved by wrapping of the DNA around gyrase, which involves the C-terminal domains (CTDs) of both GyrA subunits in the A2B2 heterotetramer. Gyrase lacking both CTDs cannot introduce negative supercoils into DNA. Here, we analyze the requirements for the two CTDs in individual steps in the supercoiling reaction. Gyrase that contains a single CTD binds, distorts, and cleaves DNA similarly to wildtype gyrase. It also shows wildtype-like DNA-dependent ATPase activity, and undergoes DNA-induced movement of the CTD as well as N-gate narrowing. Most importantly, the enzyme still introduces negative supercoils into DNA in an ATP-dependent reaction, with a velocity similar to wildtype gyrase, and decreases the linking number of the DNA in steps of two. One CTD is thus sufficient to support DNA supercoiling.

Binding and Hydrolysis of a Single ATP Is Sufficient for N-Gate Closure and DNA Supercoiling by Gyrase.

Topoisomerases catalyze the relaxation, supercoiling, catenation, and decatenation of DNA. Gyrase is a bacterial topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. The enzyme consists of two GyrB subunits, containing the ATPase domains, and two GyrA subunits. Nucleotide binding to gyrase B GyrB causes closing of the N-gate in gyrase, which orients bound DNA for supercoiling. N-gate re-opening after ATP hydrolysis, at the end of the supercoiling reaction, resets the enzyme for subsequent catalytic cycles. Gyrase binds and hydrolyzes two ATP molecules per catalytic cycle. Here, we dissect the role of these two binding and hydrolysis events using gyrase with one ATP-binding- and hydrolysis-deficient subunit, or with one binding-competent, but hydrolysis-deficient ATPase domain. We show that binding of a single ATP molecule induces N-gate closure. Gyrase that can only bind and hydrolyze a single ATP undergoes opening and closing of the N-gate in synchrony with ATP hydrolysis, and promotes DNA supercoiling under catalytic conditions. In contrast, gyrase that can bind two ATP molecules, but hydrolyzes only one, only supercoils DNA under stoichiometric conditions. Here, ATP bound to the hydrolysis-deficient subunit keeps the N-gate closed after hydrolysis of the other ATP and prevents further turnovers. Gyrase with only one functional ATPase domain hydrolyzes ATP with a similar rate to wild-type, but its supercoiling efficiency is reduced. Binding and hydrolysis of the second ATP may thus ensure efficient coupling of the nucleotide cycle with the supercoiling reaction by stabilizing the closed N-gate and by acting as a timer for N-gate re-opening.

Allosteric regulation of helicase core activities of the DEAD-box helicase YxiN by RNA binding to its RNA recognition motif.

DEAD-box proteins share a structurally similar core of two RecA-like domains (RecA_N and RecA_C) that contain the conserved motifs for ATP-dependent RNA unwinding. In many DEAD-box proteins the helicase core is flanked by ancillary domains. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA recognition motif (RRM), we investigated the effect of RNA binding to the RRM on its position relative to the core, and on core activities. RRM/RNA complex formation substantially shifts the RRM from a position close to the RecA_C to the proximity of RecA_N, independent of RNA contacts with the core. RNA binding to the RRM is communicated to the core, and stimulates ATP hydrolysis and RNA unwinding. The conformational space of the core depends on the identity of the RRM-bound RNA. Allosteric regulation of core activities by RNA-induced movement of ancillary domains may constitute a general regulatory mechanism of DEAD-box protein activity.

eIF4B stimulates eIF4A ATPase and unwinding activities by direct interaction through its 7-repeats region.

Eukaryotic translation initiation starts with binding of the eIF4F complex to the 5'-m7G cap of the mRNA. Recruitment of the 43S pre-initiation complex (PIC), formed by the 40S ribosomal subunit and other translation initiation factors, leads to formation of the 48S PIC that then scans the 5'-untranslated region (5'-UTR) toward the start codon. The eIF4F complex consists of eIF4E, the cap binding protein, eIF4A, a DEAD-box RNA helicase that is believed to unwind secondary structures in the 5'-UTR during scanning, and eIF4G, a scaffold protein that binds to both eIF4E and eIF4A. The ATPase and helicase activities of eIF4A are jointly stimulated by eIF4G and the translation initiation factor eIF4B. Yeast eIF4B mediates recruitment of the 43S PIC to the cap-bound eIF4F complex by interacting with the 40S subunit and possibly with eIF4A. However, a direct interaction between yeast eIF4A and eIF4B has not been demonstrated yet. Here we show that eIF4B binds to eIF4A in the presence of RNA and ADPNP, independent of the presence of eIF4G. A stretch of seven moderately conserved repeats, the r1-7 region, is responsible for complex formation, for modulation of the conformational energy landscape of eIF4A by eIF4B, and for stimulating the RNA-dependent ATPase- and ATP-dependent RNA unwinding activities of eIF4A. The isolated r1-7 region only slightly stimulates eIF4A conformational changes and activities, suggesting that communication of the repeats with other regions of eIF4B is required for full stimulation of eIF4A activity, for recruitment of the PIC to the mRNA and for translation initiation.

Nickel quercetinase, a "promiscuous" metalloenzyme: metal incorporation and metal ligand substitution studies.

BACKGROUND: Quercetinases are metal-dependent dioxygenases of the cupin superfamily. While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni(2+) and some other divalent metal ions. This raises the questions of which factors determine metal selection, and which metal ion is physiologically relevant. RESULTS: Metal occupancies of heterologously produced QueD proteins followed the order Ni > Co > Fe > Mn. Iron, in contrast to the other metals, does not support catalytic activity. QueD isolated from the wild-type Streptomyces sp. strain FLA contained mainly nickel and zinc. In vitro synthesis of QueD in a cell-free transcription-translation system yielded catalytically active protein when Ni(2+) was present, and comparison of the circular dichroism spectra of in vitro produced proteins suggested that Ni(2+) ions support correct folding. Replacement of individual amino acids of the 3His/1Glu metal binding motif by alanine drastically reduced or abolished quercetinase activity and affected its structural integrity. Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual catalytic activity. CONCLUSIONS: Heterologous formation of catalytically active, native QueD holoenzyme requires Ni(2+), Co(2+) or Mn(2+), i.e., metal ions that prefer an octahedral coordination geometry, and an intact 3His/1Glu motif or a 4His environment of the metal. The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance. Ni(2+) most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA.

When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding.

DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

Fluorescence methods in the investigation of the DEAD-box helicase mechanism.

DEAD-box proteins catalyze the ATP-dependent unwinding of RNA duplexes and accompany RNA molecules throughout their cellular life. Conformational changes in the helicase core of DEAD-box proteins are intimately linked to duplex unwinding. In the absence of ligands, the two RecA domains of the helicase core are separated. ATP and RNA binding induces a closure of the cleft between the RecA domains that is coupled to the distortion of bound RNA, leading to duplex destabilization and dissociation of one RNA strand. Reopening of the helicase core occurs after ATP hydrolysis and is coupled to phosphate release and dissociation of the second RNA strand.Fluorescence spectroscopy provides an array of approaches to study intermolecular interactions, local structural rearrangements, or large conformational changes of biomolecules. The fluorescence intensity of a fluorophore reports on its environment, and fluorescence anisotropy reflects the size of the molecular entity the fluorophore is part of. Fluorescence intensity and anisotropy are therefore sensitive probes to report on binding and dissociation events. Fluorescence resonance energy transfer (FRET) reports on the distance between two fluorophores and thus on conformational changes. Single-molecule FRET experiments reveal the distribution of conformational states and the kinetics of their interconversion. This chapter summarizes fluorescence approaches for monitoring individual aspects of DEAD-box protein activity, from nucleotide and RNA binding and RNA unwinding to protein and RNA conformational changes in the catalytic cycle, and illustrates exemplarily how fluorescence-based methods have contributed to understanding the mechanism of DEAD-box helicase-catalyzed RNA unwinding.

DEAD-box RNA helicase domains exhibit a continuum between complete functional independence and high thermodynamic coupling in nucleotide and RNA duplex recognition.

DEAD-box helicases catalyze the non-processive unwinding of double-stranded RNA (dsRNA) at the expense of adenosine triphosphate (ATP) hydrolysis. Nucleotide and RNA binding and unwinding are mediated by the RecA domains of the helicase core, but their cooperation in these processes remains poorly understood. We therefore investigated dsRNA and nucleotide binding by the helicase cores and the isolated N- and C-terminal RecA domains (RecA_N, RecA_C) of the DEAD-box proteins Hera and YxiN by steady-state and time-resolved fluorescence methods. Both helicases bind nucleotides predominantly via RecA_N, in agreement with previous studies on Mss116, and with a universal, modular function of RecA_N in nucleotide recognition. In contrast, dsRNA recognition is different: Hera interacts with dsRNA in the absence of nucleotide, involving both RecA domains, whereas for YxiN neither RecA_N nor RecA_C binds dsRNA, and the complete core only interacts with dsRNA after nucleotide has been bound. DEAD-box proteins thus cover a continuum from complete functional independence of their domains, exemplified by Mss116, to various degrees of inter-domain cooperation in dsRNA binding. The different degrees of domain communication and of thermodynamic linkage between dsRNA and nucleotide binding have important implications on the mechanism of dsRNA unwinding, and may help direct RNA helicases to their respective cellular processes.

eIF4B, eIF4G and RNA regulate eIF4A activity in translation initiation by modulating the eIF4A conformational cycle.

Eukaryotic translation initiation factor eIF4A is a DEAD-box helicase that resolves secondary structure elements in the 5'-UTR of mRNAs during ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. DEAD-box proteins alternate between open and closed conformations during RNA unwinding. The transition to the closed conformation is linked to duplex destabilization. eIF4A is a special DEAD-box protein that can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by the translation initiation factor eIF4G and a closed state in the presence of eIF4G and eIF4B. We show here that eIF4A alone does not measurably sample the closed conformation. The translation initiation factors eIF4B and eIF4G accelerate the eIF4A conformational cycle. eIF4G increases the rate of closing more than the opening rate, and eIF4B selectively increases the closing rate. Strikingly, the rate constants and the effect of eIF4B are different for different RNAs, and are related to the presence of single-stranded regions. Modulating the kinetics of the eIF4A conformational cycle is thus central for the multi-layered regulation of its activity, and for its role as a regulatory hub in translation initiation.

The acidic C-terminal tail of the GyrA subunit moderates the DNA supercoiling activity of Bacillus subtilis gyrase.

Gyrase is a type II DNA topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. It consists of a topoisomerase core, formed by the N-terminal domains of the two GyrA subunits and by the two GyrB subunits, that catalyzes double-stranded DNA cleavage and passage of a second double-stranded DNA through the gap in the first. The C-terminal domains (CTDs) of the GyrA subunits form a ß-pinwheel and bind DNA around their positively charged perimeter. As a result, DNA is bound as a positive supercoil that is converted into a negative supercoil by strand passage. The CTDs contain a conserved 7-amino acid motif that connects blades 1 and 6 of the ß-pinwheel and is a hallmark feature of gyrases. Deletion of this so-called GyrA-box abrogates DNA bending by the CTDs and DNA-induced narrowing of the N-gate, affects T-segment presentation, reduces the coupling of DNA binding to ATP hydrolysis, and leads to supercoiling deficiency. Recently, a severe loss of supercoiling activity of Escherichia coli gyrase upon deletion of the non-conserved acidic C-terminal tail (C-tail) of the CTDs has been reported. We show here that, in contrast to E. coli gyrase, the C-tail is a very moderate negative regulator of Bacillus subtilis gyrase activity. The C-tail reduces the degree of DNA bending by the CTDs but has no effect on DNA-induced conformational changes of gyrase that precede strand passage and reduces DNA-stimulated ATPase and DNA supercoiling activities only 2-fold. Our results are in agreement with species-specific, differential regulatory effects of the C-tail in gyrases from different organisms.

Differential contributions of the latch in Thermotoga maritima reverse gyrase to the binding of single-stranded DNA before and after ATP hydrolysis.

Reverse gyrase catalyzes the ATP-dependent introduction of positive supercoils into DNA. Supercoiling requires the functional cooperation of its N-terminal helicase domain with the C-terminal topoisomerase domain. The helicase domain contains a superfamily 2 helicase core formed by two RecA domains, H1 and H2. We show here that a helicase domain lacking the latch, an insertion in H2, fails to close the cleft in the helicase core in response to nucleotide and DNA binding at the beginning of the catalytic cycle. In the presence of the pre-hydrolysis ATP analog ADP·BeFx, however, the closed conformer can still be formed in the absence of the latch. The helicase domain lacking the latch exhibits reduced DNA affinities. The energetic difference between the two nucleotide states involved in duplex separation is diminished, rationalizing the unwinding deficiency of reverse gyrase lacking the latch. The latch most strongly contributes to binding of single-stranded DNA in the post-hydrolysis state, before phosphate release. Our results are in line with contributions of the latch in determining the direction of strand passage, and in orienting the cleaved single-stranded DNA for re-ligation. At the same time, the latch may coordinate the re-ligation reaction with strand passage and with the nucleotide cycle.

eIF4B and eIF4G jointly stimulate eIF4A ATPase and unwinding activities by modulation of the eIF4A conformational cycle.

Eukaryotic translation initiation factor 4A (eIF4A) is a DEAD-box protein that participates in translation initiation. As an ATP-dependent RNA helicase, it is thought to resolve secondary structure elements from the 5'-untranslated region of mRNAs to enable ribosome scanning. The RNA-stimulated ATPase and ATP-dependent helicase activities of eIF4A are enhanced by auxiliary proteins, but the underlying mechanisms are still largely unknown. Here, we have dissected the effect of eIF4B and eIF4G on eIF4A RNA-dependent ATPase- and RNA helicase activities and on eIF4A conformation. We show for the first time that yeast eIF4B, like its mammalian counterpart, can stimulate RNA unwinding by eIF4A, although it does not affect the eIF4A conformation. The eIF4G middle domain enhances this stimulatory effect and promotes the formation of a closed eIF4A conformation in the presence of ATP and RNA. The closed state of eIF4A has been inferred but has not been observed experimentally before. eIF4B and eIF4G jointly stimulate ATP hydrolysis and RNA unwinding by eIF4A and favor the formation of the closed eIF4A conformer. Our results reveal distinct functions of eIF4B and eIF4G in synergistically stimulating the eIF4A helicase activity in the mRNA scanning process.

Reverse gyrase transiently unwinds double-stranded DNA in an ATP-dependent reaction.

Reverse gyrase is a unique DNA topoisomerase that catalyzes the introduction of positive supercoils into DNA in an ATP-dependent reaction. It consists of a helicase domain that functionally cooperates with a topoisomerase domain. Different models for the catalytic mechanism of reverse gyrase that predict a central role of the helicase domain have been put forward. The helicase domain acts as a nucleotide-dependent conformational switch that alternates between open and closed states with different affinities for single- and double-stranded DNA. It has been suggested that the helicase domain can unwind double-stranded regions, but helicase activity has not been demonstrated as yet. Here, we show that the isolated helicase domain and full-length reverse gyrase can transiently unwind double-stranded regions in an ATP-dependent reaction. The latch region of reverse gyrase, an insertion into the helicase domain, is required for DNA supercoiling. Strikingly, the helicase domain lacking the latch cannot unwind DNA, linking unwinding to DNA supercoiling. The unwinding activity may provide and stabilize the single-stranded regions required for strand passage by the topoisomerase domain, either de novo or by expanding already existing unpaired regions that may form at high temperatures.

The GyrA-box determines the geometry of DNA bound to gyrase and couples DNA binding to the nucleotide cycle.

DNA gyrase catalyses the adenosine triphosphate-dependent introduction of negative supercoils into DNA. The enzyme binds a DNA-segment at the so-called DNA-gate and cleaves both DNA strands. DNA extending from the DNA-gate is bound at the perimeter of the cylindrical C-terminal domains (CTDs) of the GyrA subunit. The CTDs are believed to contribute to the wrapping of DNA around gyrase in a positive node as a prerequisite for strand passage towards negative supercoiling. A conserved seven amino acid sequence motif in the CTD, the so-called GyrA-box, has been identified as a hallmark feature of gyrases. Mutations of the GyrA-box abolish supercoiling. We show here that the GyrA-box marginally stabilizes the CTDs. Although it does not contribute to DNA binding, it is required for DNA bending and wrapping, and it determines the geometry of the bound DNA. Mutations of the GyrA-box abrogate a DNA-induced conformational change of the gyrase N-gate and uncouple DNA binding and adenosine triphosphate hydrolysis. Our results implicate the GyrA-box in coordinating DNA binding and the nucleotide cycle.