Formation of a nucleoprotein complex containing Tn7 and its target DNA regulates transposition initiation

Tn7 insertion into its specific target site, attTn7, is mediated by the proteins TnsA, TnsB, TnsC and TnsD. The double-strand breaks that separate Tn7 from the donor DNA require the Tns proteins, the transposon and an attTn7 target DNA, suggesting that a prerequisite for transposition is the formation of a nucleoprotein complex containing TnsABC+D, and these DNAs. Here, we identify a TnsABC+D transposon-attTn7 complex, and demonstrate that it is a transposition intermediate. We demonstrate that an interaction between TnsB, the transposase subunit that binds to the transposon ends, and TnsC, the target DNA-binding protein that controls the activity of the transposase, is essential for assembly of the TnsABC+D transposon-attTn7 complex. We also show that certain TnsB residues are required for recombination because they mediate a TnsB-TnsC interaction critical to formation of the TnsABC+D transposon-attTn7 complex. We demonstrate that TnsA, the other transposase subunit, which also interacts with TnsC, greatly stabilizes the TnsABC+D transposon-attTn7 complex. Thus multiple interactions between the transposase subunits, TnsA and TnsB, and the target-binding transposase activator, TnsC, control Tn7 transposition.     

Avoiding self: two Tn7-encoded proteins mediate target immunity in Tn7 transposition.

The bacterial transposon Tn7 exhibits target immunity, a process that prevents Tn7 from transposing into target DNAs that already contain a copy of the transposon. This work investigates the mechanism of target immunity in vitro. We demonstrate that two Tn7-encoded proteins_TnsB, which binds specifically to the ends of Tn7, and TnsC, the ATP-dependent DNA binding protein_act as a molecular switch to impose immunity on target DNAs containing Tn7 (or just Tn7 ends). TnsC binds to target DNA molecules and communicates with the Tn7 transposition machinery; here we show that target DNAs containing Tn7 ends are also bound and subsequently inactivated by TnsB. Protein-protein interactions between TnsB and TnsC appear to be responsible for this inactivation; the target DNA promotes these interactions by tethering TnsB and TnsC in high local concentration. An attractive model that emerges from this work is that TnsB triggers the dissociation of TnsC from the Tn7 end-containing target DNA; that dissociation depends on TnsC's ability to hydrolyze ATP. We propose that these interactions between TnsB and TnsC not only prevent Tn7 from inserting into itself, but also facilitate the selection of preferred target sites that is the hallmark of Tn7 transposition.     

Isolation and characterization of Tn7 transposase gain-of-function mutants: a model for transposase activation

Tn7 transposition has been hypothesized to require a heteromeric transposase formed by two Tn7-encoded proteins, TnsA and TnsB, and accessory proteins that activate the transposase when they are associated with an appropriate target DNA. This study investigates the mechanism of Tn7 transposase activation by isolation and analysis of transposase gain-of-function mutants that are active in the absence of these accessory proteins. This work shows directly that TnsA and TnsB are essential and sufficient components of the Tn7 transposase and also provides insight into the signals that activate the transposase. We also describe a protein-protein interaction between TnsA and TnsC, a regulatory accessory protein, that is likely to be critical for transposase activation.     

Analysis of gain-of-function mutants of an ATP-dependent regulator of Tn7 transposition

The bacterial transposon Tn7 is distinguished by its unusual discrimination among targets, being particularly attracted to certain target DNA and actively avoiding other DNA. Tn7 transposition is mediated by the interaction of two alternative transposon-encoded target selection proteins, TnsD and TnsE, with a common core transposition machinery composed of the transposase (TnsAB) and an ATP-dependent DNA-binding protein TnsC. No transposition is observed with wild-type TnsABC. Here, we analyze the properties of two gain-of-function TnsC mutants that allow transposition in the absence of TnsD or TnsE. We find that these TnsC mutants have altered interactions with ATP and DNA that can account for their gain-of-function phenotype. We also show that TnsC is an ATPase and that it directly interacts with the TnsAB transposase. This work provides strong support to the view that TnsC and its ATP state are central to the control of Tn7 transposition.     

Target DNA structure plays a critical role in Tn7 transposition

The bacterial transposon Tn7 utilizes four Tn7-encoded proteins, TnsA, TnsB, TnsC and TnsD, to make insertions at a specific site termed attTn7. This target is selected by the binding of TnsD to attTn7 in a sequence-specific manner, followed by the binding of TnsC and activation of the transposase. We show that TnsD binding to attTn7 induces a distortion at the 5' end of the binding site and TnsC contacts the region of attTn7 distorted by TnsD. Previous work has shown that a target site containing triplex DNA, instead of TnsD-attTn7, can recruit TnsABC and effect site- specific insertion of Tn7. We propose that the DNA distortion imposed by TnsD on attTn7, like the altered DNA structure via triplex formation, serves as a signal to recruit TnsC. We also show that TnsD primarily contacts the major groove of DNA, whereas TnsC is a minor groove binding protein. The footprint of the TnsC-TnsD-attTn7 nucleoprotein complex includes and extends beyond the Tn7 insertion site, where TnsC forms a platform to receive and activate the transposase to carry out recombination.     

Architecture of the Tn7 Posttransposition Complex: An Elaborate Nucleoprotein Structure

Four transposition proteins encoded by the bacterial transposon Tn7, TnsA, TnsB, TnsC, and TnsD, mediate its site- and orientation-specific insertion into the chromosomal site attTn7. To establish which Tns proteins are actually present in the transpososome that executes DNA breakage and joining, we have determined the proteins present in the nucleoprotein product of transposition, the posttransposition complex (PTC), using fluorescently labeled Tns proteins. All four required Tns proteins are present in the PTC in which we also find that the Tn7 ends are paired by protein-protein contacts between Tns proteins bound to the ends. Quantification of the relative amounts of the fluorescent Tns proteins in the PTC indicates that oligomers of TnsA, TnsB, and TnsC mediate Tn7 transposition. High-resolution DNA footprinting of the DNA product of transposition attTn7∷Tn7 revealed that about 350 bp of DNA on the transposon ends and on attTn7 contact the Tns proteins. All seven binding sites for TnsB, the component of the transposase that specifically binds the ends and mediates 3′ end breakage and joining, are occupied in the PTC. However, the protection pattern of the sites closest to the Tn7 ends in the PTC are different from that observed with TnsB alone, likely reflecting the pairing of the ends and their interaction with the target nucleoprotein complex necessary for activation of the breakage and joining steps. We also observe extensive protection of the attTn7 sequences in the PTC and that alternative DNA structures in substrate attTn7 that are imposed by TnsD are maintained in the PTC.     

The carboxy-terminal portion of TnsC activates the Tn7 transposase through a specific interaction with TnsA

Tn7 transposition requires the assembly of a nucleoprotein complex containing four self-encoded proteins, transposon ends, and target DNA. Within this complex, TnsC, the molecular switch that regulates transposition, and TnsA, one part of the transposase, interact directly. Here, we demonstrate that residues 504-555 of TnsC are responsible for TnsA/TnsC interaction. The crystal structure of the TnsA/TnsC(504-555) complex, resolved to 1.85 A, illustrates the burial of a large hydrophobic patch on the surface of TnsA. One consequence of sequestering this patch is a marked increase in the thermal stability of TnsA as shown by differential scanning calorimetry. A model based on the complex structure suggested that TnsA and a slightly longer version of the cocrystallized TnsC fragment (residues 495-555) might cooperate to bind DNA, a prediction confirmed using gel mobility shift assays. Donor DNA binding by the TnsA/TnsC(495-555) complex is correlated with the activation of the TnsAB transposase, as measured by double-stranded DNA cleavage assays, demonstrating the importance of the TnsA/TnsC interaction in affecting Tn7 transposition.     

A minimal system for Tn7 transposition: the transposon-encoded proteins TnsA and TnsB can execute DNA breakage and joining reactions that generate circularized Tn7 species

In the presence of ATP and Mg(2+), the bacterial transposon Tn7 translocates via a cut and paste mechanism executed by the transposon-encoded proteins TnsA+TnsB+TnsC+TnsD. We report here that in the presence of Mn(2+), TnsA+TnsB alone can execute the DNA breakage and joining reactions of Tn7 recombination. ATP is not essential in this minimal system, revealing that this cofactor is not directly involved in the chemical steps of recombination. In both the TnsAB and TnsABC+D systems, recombination initiates with double-strand breaks at each transposon end that cut Tn7 away from flanking donor DNA. In the minimal system, breakage occurs predominantly at a single transposon end and the subsequent end-joining reactions are intramolecular, with the exposed 3' termini of a broken transposon end joining near the other end of the Tn7 element in the same donor molecule to form circular transposon species. In contrast, in TnsABC+D recombination, breaks occur at both ends of Tn7 and the two ends join to a target site on a different DNA molecule to form an intermolecular simple insertion. This demonstration of the capacity of TnsAB to execute breakage and joining reactions supports the view that these proteins form the Tn7 transposase.     

The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products.

The bacterial transposon Tn7 translocates by a cut and paste mechanism: excision from the donor site results from double-strand breaks at each end of Tn7 and target insertion results from joining of the exposed 3'' Tn7 tips to the target DNA. Through site-directed mutagenesis of the Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate that the Tn7 transposase is a heteromeric complex of these proteins, each protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5'' ends of Tn7, and TnsB mediates DNA breakage and joining reactions at the 3'' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result from a collaboration between two active sites, one in TnsA and one in TnsB; the same (or a closely related) active site in TnsB also mediates the subsequent joining of the 3'' ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative DD(35)E motifs blocks catalytic activity. Recombinases of this class require a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a TnsA mutant containing a sulfur (cysteine) substitution, we provide evidence that a divalent metal actually interacts with these acidic amino acids.     

Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA.

The bacterial transposon Tn7 encodes five transposition genes tnsABCDE. We report a simple and rapid procedure for the purification of TnsC protein. We show that purified TnsC is active in and required for Tn7 transposition in a cell-free recombination system. This finding demonstrates that TnsC participates directly in Tn7 transposition and explains the requirement for tnsC function in Tn7 transposition. We have found that TnsC binds adenine nucleotides and is thus a likely site of action of the essential ATP cofactor in Tn7 transposition. We also report that TnsC binds non-specifically to DNA in the presence of ATP or the generally non-hydrolyzable analogues AMP-PNP and ATP-gamma-S, and that TnsC displays little affinity for DNA in the presence of ADP. We speculate that TnsC plays a central role in the selection of target DNA during Tn7 transposition.     

Gain-of-Function Mutations in Tnsc, an Atp-Dependent Transposition Protein That Activates the Bacterial Transposon Tn7

The bacterial transposon Tn7 encodes five genes whose protein products are used in different combinations to direct transposition to different types of target sites. TnsABC+D directs transposition to a specific site in the Escherichia coli chromosome called attTn7, whereas TnsABC+E directs transposition to non-attTn7 sites. These transposition reactions can also recognize and avoid ``immune' targets that already contain a copy of Tn7. TnsD and TnsE are required to activate TnsABC as well as to select a target site; no transposition occurs with wild-type TnsABC alone. Here, we describe the isolation of TnsC gain-of-function mutants that activate the TnsA+B transposase in the absence of TnsD or TnsE. Some of these TnsC mutants enable the TnsABC machinery to execute transposition without sacrificing its ability to discriminate between different types of targets. Other TnsC mutants appear to constitutively activate the TnsABC machinery so that it bypasses target signals. We also present experiments that suggest that target selection occurs early in the Tn7 transposition pathway in vivo: favorable attTn7 targets appear to promote the excision of Tn7 from the chromosome, whereas immune targets do not allow transposon excision to occur. This work supports the view that TnsC plays a central role in the evaluation and utilization of target DNAs.     

Interaction of the Tn7-encoded transposition protein TnsB with the ends of the transposon.

We have used several high resolution methods to examine the interaction of TnsB, a transposition protein encoded by the bacterial transposon Tn7, with its binding sites at the ends of the transposon. These binding sites lie within the DNA segments that are directly involved in transposition. We show that the binding of TnsB to DNA can promote DNA bending, suggesting that the interaction of TnsB with the ends may result in formation of a highly organized protein-DNA complex. We also identify likely positions of close contact between of TnsB and its binding sites. Analysis of the interaction of TnsB with intact Tn7 ends reveals TnsB occupies its binding sites in a particular order, the sites immediately adjacent to the transposon termini being occupied only after other inner sites are bound. Such ordered occupancy suggests that the various binding sites have differing apparent affinities for TnsB.     

Purification of TnsB, a transposition protein that binds to the ends of Tn7.

We have purified TnsB, a transposition protein encoded by the bacterial transposon Tn7. The purification procedure involves three chromatographic steps (DNA-cellulose, norleucine-Sepharose, and phosphocellulose) and yields milligram quantities of highly purified protein. The apparent molecular mass of denatured TnsB protein is approximately 85 kDa. Gel filtration chromatography and sucrose gradient sedimentation studies indicate that in solution, native TnsB is a monomer of nonspherical shape. Using DNase I protection analysis, we established that TnsB is a sequence-specific DNA-binding protein that recognizes multiple sites in both ends of the transposon. The TnsB binding sites, three in the left end of Tn7 and four in the right end, are highly related in nucleotide sequence and are located in DNA segments that we have previously shown contain cis-acting sequences important for Tn7 transposition. Our results also show that one of the TnsB binding sites overlaps a proposed promoter for the transposition genes of Tn7. These studies suggest that the specific binding of TnsB to the ends of Tn7 mediates recombination and may also regulate the expression of Tn7-encoded transposition genes.     

Identification of transposition proteins encoded by the bacterial transposon Tn7.

The bacterial transposon, Tn7, encodes an elaborate array of transposition genes, tnsABCDE. We report here the direct identification of the TnsA, TnsB, TnsC and TnsD polypeptides by immunoblotting. Our results demonstrate that the complexity of the protein information devoted to Tn7 transposition is considerable: the aggregate molecular size of the five Tns polypeptides is about 300 kDa. We also report the sequence of the tnsA gene and of the 5' ends of tnsB and tnsD. This analysis reveals that all five tns genes are oriented in the same direction within Tn7.     

Selective recognition of pyrimidine motif triplexes by a protein encoded by the bacterial transposon Tn7

The bacterial transposon Tn7 is distinguished among mobile genetic elements by its targeting abilities. Recently, we reported that Tn7 is able to selectively insert adjacent to triple-helical DNA. The binding of TnsC, a Tn7-encoded protein, to the triplex DNA target leads to the specific transposition of Tn7 adjacent to both inter- and intramolecular pyrimidine motif triplexes. Here, we further probe how Tn7 targets triplex DNA. We report that TnsC discriminates between different types of triplexes, showing binding preference for pyrimidine but not for purine motif intermolecular triplex DNA. The binding preferences of TnsC and the Tn7 insertion profiles were obtained using psoralenated, triplex- forming oligonucleotides annealed to plasmid DNAs. Although the presence of psoralen is not required for targeting nor is it alone able to attract TnsC, we show that the location of psoralen within the pyrimidine motif triplex does alter the position of Tn7 insertion relative to the triplex. Comparison between the triplex-targeting pathway and the highly site-specific targeting pathway mediated by the binding of the Tn7-encoded protein, TnsD, to the unique site attTn7, suggests that similar structural features within each target DNA are recognized by TnsC, leading to site-specific transposition. This work demonstrates that a prokaryotic protein involved in the targeting and regulation of Tn7 translocation, TnsC, can selectively recognize pyrimidine motif triplexes.     

Host proteins can stimulate Tn7 transposition: a novel role for the ribosomal protein L29 and the acyl carrier protein.

The bacterial transposon Tn7 is distinguished by its ability to insert at a high frequency into a specific site in the Escherichia coli chromosome called attTn7. Tn7 insertion into attTn7 requires four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC and TnsD. The selection of attTn7 is determined by TnsD, a sequence-specific DNA-binding protein. TnsD binds attTn7 and interacts with TnsABC, the core transposition machinery, which facilitates the insertion of Tn7 into attTn7. In this work, we report the identification of two host proteins, the ribosomal protein L29 and the acyl carrier protein (ACP), which together stimulate the binding of TnsD to attTn7. The combination of L29 and ACP also stimulates Tn7 transposition in vitro. Interestingly, mutations in L29 drastically decrease Tn7 transposition in vivo, and this effect of L29 on Tn7 transposition is specific for TnsABC+D reactions.     

Purification and characterisation of the TnsB protein of Tn7: a transposition protein that binds to the ends of Tn7.

Tn7, a large bacterial transposon encodes 5 proteins required for its transposition. We report a rapid and easy purification of one of these proteins, TnsB, from an overexpression strain. This protein was shown to bind to the ends of Tn7, in a bandshift assay, in two distinct stages as a function of protein concentration. DNasel footprinting at each end of Tn7 showed that the TnsB recognition sequence, a set of 22 bp repeats, plus Tn7 termini are protected. Binding of TnsB appeared cooperative but was only observed above a threshold concentration of protein. ATP and Mg2+ had no effect on the pattern of protection, nor did addition of other Tn7-encoded proteins. Hydroxyl radical footprinting, performed at the right end, showed that TnsB binds preferentially to one side of the DNA helix.     

Tn7: a target site-specific transposon

The bacterial transposon Tn7 is an unusual mobile DNA segment. Most transposable elements move at low-frequency and display little target site-selectivity. By contrast, Tn7 inserts at high-frequency into a single specific site in the chromosomes of many bacteria. In the absence of this specific site, called attTn7 in Escherichia coli where Tn7 has been most extensively studied, Tn7 transposes at low-frequency and inserts into many different sites. Much has recently been learned about Tn7 transposition from both genetic and biochemical studies. The Tn7 recombination machinery is elaborate and includes a large number of Tn7-encoded proteins, probably host-encoded proteins and also rather large cis-acting transposition sequences at the transposon termini and at the target site. Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.     

Separate structural and functional domains of Tn4430 transposase contribute to target immunity

Like other transposons of the Tn3 family, Tn4430 exhibits target immunity, a process that prevents multiple insertions of the transposon into the same DNA molecule. Immunity is conferred by the terminal inverted repeats of the transposon and is specific to each element of the family, indicating that the transposase TnpA is directly involved in the process.However, the molecular mechanism whereby this protein promotes efficient transposition into permissive targets while preventing transposition into immune targets remains unknown. Here, we demonstrate that both functions of TnpA can be uncoupled from each other by isolating and characterizing mutants that are proficient in transposition (T+) but impaired in immunity (I-). The identified T+/I- mutations are clustered into separate structural and functional domains of TnpA, indicating that different activities of the protein contribute to immunity.Combination of separate mutations had synergistic effects on target immunity but contrasting effects on transposition. One class of mutations was found to stimulate transposition, whereas other mutations appeared to reduce TnpA activity. The data are discussed with respect to alternative models in which TnpA acts as a specific determinant to both establish and respond to immunity.     

Tn5 as a model for understanding DNA transposition

Tn5 is an excellent model system for understanding the molecular basis of DNA-mediated transposition. Mechanistic information has come from genetic and biochemical investigations of the transposase and its interactions with the recognition DNA sequences at the ends of the transposon. More recently, molecular structure analyses of catalytically active transposase; transposon DNA complexes have provided us with unprecedented insights into this transposition system. Transposase initiates transposition by forming a dimeric transposase, transposon DNA complex. In the context of this complex, the transposase then catalyses four phosphoryl transfer reactions (DNA nicking, DNA hairpin formation, hairpin resolution and strand transfer into target DNA) resulting in the integration of the transposon into its new DNA site. The studies that elucidated these steps also provided important insights into the integration of retroviral genomes into host DNA and the immune system V(D)J joining process. This review will describe the structures and steps involved in Tn5 transposition and point out a biologically important although surprising characteristic of the wild-type Tn5 transposase. Transposase is a very inactive protein. An inactive transposase protein ensures the survival of the host and thus the survival of Tn5.