Tn5 transposition: a molecular tool for studying protein structure-function.

Transposon-based technologies are important genetic tools for global genome analysis and, as discussed in the present paper, in detailed studies of protein structure-function. Various different transposition systems can be used in these studies but this paper uses Tn5-related systems as a model. In particular, the following four different technologies are described in this paper: (i) using transposition to generate nested deletion families, (ii) using transposons to generate functional protein fusions to reporter functions, (iii) mapping protein secondary structures through the generation and analysis of in-frame linker insertions and (iv) using sequential transposition events to generate random gene fusions. The success of these forward genetic technologies requires that the transposition system be efficient and manifest near-random target sequence selection.

Functional characterization of arginine 30, lysine 40, and arginine 62 in Tn5 transposase.

Three N-terminal basic residues of Tn5 transposase, which are associated with proteolytic cleavages by Escherichia coli proteinases, were mutated to glutamine residues with the goal of producing more stable transposase molecules. Mutation of either arginine 30 or arginine 62 to glutamine produced transposase molecules that were more stable toward E. coli proteinases than the parent hyperactive Tn5 transposase, however, they were inactive in vivo. In vitro analysis revealed these mutants were inactive, because both Arg(30) and Arg(62) are required for formation of the paired ends complexes when the transposon is attached to the donor backbone. These results suggest Arg(30) and Arg(62) play critical roles in DNA binding and/or synaptic complex formation. Mutation of lysine 40 to glutamine did not increase the overall stability of the transposase to E. coli proteinases. This mutant transposase was only about 1% as active as the parent hyperactive transposase in vivo; however, it retained nearly full activity in vitro. These results suggest that lysine 40 is important for a step in the transposition mechanism that is bypassed in the in vitro assay system, such as the removal of the transposase molecule from DNA following strand transfer.

Characterization of a Tn5 pre-cleavage synaptic complex.

Protein catalyzed DNA rearrangements typically require assembly of complex nucleoprotein structures. In transposition and integration reactions, these structures, termed synaptic complexes, are mandatory for catalysis. We characterize the Tn5 pre-cleavage synaptic complex, the simplest transposition complex described to date. We identified this complex by gel retardation assay using short, linear fragments and have shown that it contains a dimer of transposase, two DNA molecules, and is competent for DNA cleavage in the presence of Mg(2+). We also used hydroxyl radical footprinting and interference techniques to delineate the protein-DNA contacts made in the Tn5 synaptic and monomer complexes. All positions (except position 1) of the end sequence are contacted by transposase in the synaptic complex. We have determined that positions 2-5 of the end sequence are specifically required for synaptic complex formation as they are not required for monomer complex formation. In addition, in the synaptic complex, there is a strong, local distortion centered around position 1 which likely facilitates cleavage.

Trans catalysis in Tn5 transposition.

Synaptic complexes in prokaryotic transposons occur when transposase monomers bind to each of two specific end-binding sequences and then associate to bring the proteins and the two ends of the transposon together. It is within this complex of proteins and DNA that identical catalytic reactions are carried out by transposase on each of the ends of the transposon. In this study, we perform in vitro transposition reactions by combining the methylated inside end (IE(ME)) biased hyperactive Tn5 transposase, Tnp sC7 version 2.0, and the outside end (OE) biased hyperactive Tn5 transposase, Tnp EK/LP, with plasmid DNA containing a transposon defined by one IE(ME) and one OE. These two proteins cooperate to facilitate double end cleavage of the transposon from the plasmid and conversion into transposition products via strand transfer. When one of the hyperactive Tnps is replaced with a catalytically inactive version containing the mutation EA326 (DDE mutant), the predominant reaction product is a linearized plasmid resulting from single end cleavage. Restriction analysis of these linear products reveals that cleavage is occurring on the end distal to that which is bound by the transposase with an intact active site or in trans. Similar in vitro experiments performed with precut transposons and a supercoiled target plasmid demonstrated that the strand transfer reaction is also facilitated by a trans active DDE motif.

Three-dimensional structure of the Tn5 synaptic complex transposition intermediate.

Genomic evolution has been profoundly influenced by DNA transposition, a process whereby defined DNA segments move freely about the genome. Transposition is mediated by transposases, and similar events are catalyzed by retroviral integrases such as human immunodeficiency virus-1 (HIV-1) integrase. Understanding how these proteins interact with DNA is central to understanding the molecular basis of transposition. We report the three-dimensional structure of prokaryotic Tn5 transposase complexed with Tn5 transposon end DNA determined to 2.3 angstrom resolution. The molecular assembly is dimeric, where each double-stranded DNA molecule is bound by both protein subunits, orienting the transposon ends into the active sites. This structure provides a molecular framework for understanding many aspects of transposition, including the binding of transposon end DNA by one subunit and cleavage by a second, cleavage of two strands of DNA by a single active site via a hairpin intermediate, and strand transfer into target DNA.

The C-terminal alpha helix of Tn5 transposase is required for synaptic complex formation.

An important step in Tn5 transposition requires transposase-transposase homodimerization to form a synaptic complex competent for cleavage of transposon DNA free from the flanking sequence. We demonstrate that the C-terminal helix of Tn5 transposase (residues 458-468 of 476 total amino acids) is required for synaptic complex formation during Tn5 transposition. Specifically, deletion of eight amino acids or more from the C terminus greatly reduces or abolishes synaptic complex formation in vitro. Due to this impaired synaptic complex formation, transposases lacking eight amino acids are also defective in the cleavage step of transposition. Interactions within the synaptic complex dimer interface were investigated by site-directed mutagenesis, and residues required for synaptic complex formation include amino acids comprising the dimer interface in the Tn5 inhibitor x-ray crystal structure dimer. Because the crystal structure dimer was hypothesized to be the inhibitory complex and not a synaptic complex, this result was surprising. Based on these data, models for both in vivo and in vitro synaptic complex formation are presented.

Structural studies of lacUV5-RNA polymerase interactions in vitro. Ethylation interference and missing nucleoside analysis.

Substantial effort has been made to investigate the interactions that the Escherichia coli RNA polymerase makes with promoter DNA during transcription initiation. The lacUV5 promoter has been the object of many of these studies, and to date, an incredible wealth of information exists on how RNA polymerase interacts with this promoter. We have sought to expand current knowledge by the use of two chemical interference protocols, phosphate ethylation and missing nucleoside. We have added to existing information with the identification of additional phosphates, for example, at the start site of the template strand that, when ethylated, perturb the binding of RNA polymerase. We have also discovered a number of positions, most remarkably -37 to -34 of the nontemplate strand, where nucleoside loss decreases binding. Finally, we have discovered positions of ethylation and/or nucleoside loss that can stimulate binding. In particular, missing nucleosides and phosphate ethylation near the transcription start site enhance RNA polymerase binding.

Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes.

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene-operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1, and Ty1) have been described, which bypass many limitations of in vivo systems. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.

Tn5: A molecular window on transposition.

DNA transposition is an underlying process involved in the remodeling of genomes in all types of organisms. We analyze the multiple steps in cut-and-paste transposition using the bacterial transposon Tn5 as a model. This system is particularly illuminating because of the existence of structural, genetic, and biochemical information regarding the two participating specific macromolecules: the transposase and the 19-bp sequences that define the ends of the transposon. However, most of the insights should be of general interest because of similarities to other transposition-like systems such as HIV-1 DNA integration into the host genome.

Hairpin formation in Tn5 transposition.

The initial chemical steps in Tn5 transposition result in blunt end cleavage of the transposon from the donor DNA. We demonstrate that this cleavage occurs via a hairpin intermediate. The first step is a 3' hydrolytic nick by transposase. The free 3'OH then attacks the phosphodiester bond on the opposite strand, forming a hairpin at the transposon end. In addition to forming precise hairpins, Tn5 transposase can form imprecise hairpins. This is the first example of imprecise hairpin formation on transposon end DNA. To undergo strand transfer, the hairpin must to be resolved by a transposase-catalyzed hydrolytic cleavage. We show that both precise and imprecise hairpins are opened by transposase. A transposition mechanism utilizing a hairpin intermediate allows a single transposase active site to cleave both 3' and 5' strands without massive protein/DNA rearrangements.

Escherichia coli DNA topoisomerase I copurifies with Tn5 transposase, and Tn5 transposase inhibits topoisomerase I.

Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. Genetic evidence suggested that this killing involves titration of E. coli topoisomerase I (Topo I). Here, we present biochemical evidence that supports this model. Tn5 Tnp copurifies with Topo I while nonkilling derivatives of Tnp, Delta37Tnp and Delta55Tnp (Inhibitor [Inh]), show reduced affinity or no affinity, respectively, for Topo I. In agreement with these results, the presence of Tnp, but not Delta37 or Inh derivatives of Tnp, inhibits the DNA relaxation activity of Topo I in vivo as well as in vitro. Other proteins, including RNA polymerase, are also found to copurify with Tnp. For RNA polymerase, reduced copurification with Tnp is observed in extracts from a topA mutant strain, suggesting that RNA polymerase interacts with Topo I and not Tnp.

The three-dimensional structure of a Tn5 transposase-related protein determined to 2.9-A resolution.

Transposon Tn5 employs a unique means of self-regulation by expressing a truncated version of the transposase enzyme that acts as an inhibitor. The inhibitor protein differs from the full-length transposase only by the absence of the first 55 N-terminal amino acid residues. It contains the catalytic active site of transposase and a C-terminal domain involved in protein-protein interactions. The three-dimensional structure of Tn5 inhibitor determined to 2.9-A resolution is reported here. A portion of the protein fold of the catalytic core domain is similar to the folds of human immunodeficiency virus-1 integrase, avian sarcoma virus integrase, and bacteriophage Mu transposase. The Tn5 inhibitor contains an insertion that extends the beta-sheet of the catalytic core from 5 to 9 strands. All three of the conserved residues that make up the "DDE" motif of the active site are visible in the structure. An arginine residue that is strictly conserved among the IS4 family of bacterial transposases is present at the center of the active site, suggesting a catalytic motif of "DDRE." A novel C-terminal domain forms a dimer interface across a crystallographic 2-fold axis. Although this dimer represents the structure of the inhibited complex, it provides insight into the structure of the synaptic complex.

A mechanism for Tn5 inhibition. carboxyl-terminal dimerization.

Tn5 is unique among prokaryotic transposable elements in that it encodes a special inhibitor protein identical to the Tn5 transposase except lacking a short NH2-terminal DNA binding sequence. This protein regulates transposition through nonproductive protein-protein interactions with transposase. We have studied the mechanism of Tn5 inhibition in vitro and find that a heterodimeric complex between the inhibitor and transposase is critical for inhibition, probably via a DNA-bound form of transposase. Two dimerization domains are known in the inhibitor/transposase shared sequence, and we show that the COOH-terminal domain is necessary for inhibition, correlating with the ability of the inhibitor protein to homodimerize via this domain. This regulatory complex may provide clues to the structures of functional synaptic complexes. Additionally, we find that NH2- and COOH-terminal regions of transposase or inhibitor are in functional contact. The NH2 terminus appears to occlude transposase homodimerization (hypothetically mediated by the COOH terminus), an effect that might contribute to productive transposition. Conversely, a deletion of the COOH terminus uncovers a secondary DNA binding region in the inhibitor protein which is probably located near the NH2 terminus.

Escherichia coli DNA topoisomerase I and suppression of killing by Tn5 transposase overproduction: topoisomerase I modulates Tn5 transposition.

Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. The overproduction causes cell filamentation and abnormal chromosome segregation. Here we present three lines of evidence strongly suggesting that Tnp overproduction killing is due to titration of topoisomerase I. First, a suppressor mutation of transposase overproduction killing, stkD10, is localized in topA (the gene for topoisomerase I). The stkD10 mutant has the following characteristics: first, it has an increased abundance of topoisomerase I protein, the topoisomerase I is defective for the DNA relaxation activity, and DNA gyrase activity is reduced; second, the suppressor phenotype of a second mutation localized in rpoH, stkA14 (H. Yigit and W. S. Reznikoff, J. Bacteriol. 179:1704-1713, 1997), can be explained by an increase in topA expression; and third, overexpression of wild-type topA partially suppresses the killing. Finally, topoisomerase I was found to enhance Tn5 transposition up to 30-fold in vivo.

Tn5/IS50 target recognition.

This communication reports an analysis of Tn5/IS50 target site selection by using an extensive collection of Tn5 and IS50 insertions in two relatively small regions of DNA (less than 1 kb each). For both regions data were collected resulting from in vitro and in vivo transposition events. Since the data sets are consistent and transposase was the only protein present in vitro, this demonstrates that target selection is a property of only transposase. There appear to be two factors governing target selection. A target consensus sequence, which presumably reflects the target selection of individual pairs of Tn5/IS50 bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn5/IS50 target site is A-GNTYWRANC-T. However, we observed that independent insertion sites tend to form groups of closely located insertions (clusters), and insertions very often were spaced in a 5-bp periodic fashion. This suggests that Tn5/IS50 target selection is facilitated by more than two transposase protomers binding to the DNA, and, thus, for a site to be a good target, the overlapping neighboring DNA should be a good target, too. Synthetic target sequences were designed and used to test and confirm this model.

CAP, the -45 region, and RNA polymerase: three partners in transcription initiation at lacP1 in Escherichia coli.

The lac operon of Escherichia coli is positively regulated by the catabolite activator protein (CAP) bound upstream of the -45 region (CAP binding is centered at -61.5; the -45 region extends from -50 to -38). Certain mutations within the -45 region generate sequences that resemble UP elements in base composition and mimic the stimulation by the rrnBP1 UP element, yielding up to 15-fold stimulation in vivo. These -45 region "UP mutants" are compromised in their CAP stimulation. CAP and UP elements do not act in a fully additive manner in vivo at the lac operon. Transcription assays with the wild-type lac promoter and an UP mutant of lac indicate that CAP and UP DNA also fail to act in a completely additive manner in vitro. RNA polymerase can stabilize CAP binding to promoter DNA with a -45 region UP element against a heparin challenge. This shows that CAP and the UP DNA do not compete for the alpha-CTD as a mechanism for their lack of additivity. CAP and UP elements both demonstrate decreased stimulation of transcription as RNA polymerase concentration is increased from 0.05 to 10 nM in in vitro transcription experiments. In addition CAP also stimulates transcription in a manner that does not decrease as RNA polymerase is varied over this concentration range. This invariable stimulation is by two- to threefold and occurs both in vivo and in vitro. It is not dependent upon the alpha-CTD of RNA polymerase and is maintained in the presence of the AR1 CAP mutant HL159. This two- to threefold invariable CAP stimulation appears to depend on the -45 region sequence as our -45 region mutants demonstrate different responses to HL159 CAP stimulation in vivo.

Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition.

We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions. A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene. Any Amprreplicating plasmid that has undergone a transposition event (Kans) will be targeted primarily to any insert in the MCS. Two different size targets were tested in the in vitro system. Synthetic transposon plasmids containing either target were incubated in the presence of purified transposase (Tnp) protein and transformed. Transposition frequencies (Ampr/Kans) for both targets were found to be 30-50%, of which >95% occur within the target sequence, in an apparently random manner. By a conservative estimate 10(5) or more deletions/inversions within a given segment of DNA can be expected from a single one-step 20 microl transposition reaction. These nested deletions can be used for structure-function analysis of proteins and for sequence analysis. The inversions provide nested sequencing templates of the opposite strand from the deletions.

Functional characterization of the Tn5 transposase by limited proteolysis.

The 476 amino acid Tn5 transposase catalyzes DNA cutting and joining reactions that cleave the Tn5 transposon from donor DNA and integrate it into a target site. Protein-DNA and protein-protein interactions are important for this tranposition process. A truncated transposase variant, the inhibitor, decreases transposition rates via the formation of nonproductive complexes with transposase. Here, the inhibitor and the transposase are shown to have similar secondary and tertiary folding. Using limited proteolysis, the transposase has been examined structurally and functionally. A DNA binding region was localized to the N-terminal 113 amino acids. Generally, the N terminus of transposase is sensitive to proteolysis but can be protected by DNA. Two regions are predicted to contain determinants for protein-protein interactions, encompassing residues 114-314 and 441-476. The dimerization regions appear to be distinct and may have separate functions, one involved in synaptic complex formation and one involved in nonproductive multimerization. Furthermore, predicted catalytic regions are shown to lie between major areas of proteolysis.

Molecular genetic analysis of transposase-end DNA sequence recognition: cooperativity of three adjacent base-pairs in specific interaction with a mutant Tn5 transposase.

Transposition of Tn5 and IS50 requires the specific binding of transposase (Tnp) to the end inverted repeats, the outside end (OE) and the inside end (IE). OE and IE have 12 identical base-pairs and seven non-identical base-pairs. Previously we described the isolation of a Tnp mutant, EK54, that shows an altered preference for OE versus IE compared to wild-type (wt) Tnp. EK54 enhances OE recognition and decreases IE recognition both in DNA binding and in overall transposition. Here we report that base-pairs 10, 11 and 12 of the OE are critical for the specific recognition by EK54 Tnp. These three adjacent base-pairs act cooperatively; all three must be present in order for EK54 Tnp to interact very favorably with the end DNA. The existence of only one or two of these three base-pairs decreases binding of EK54 Tnp. The combined use of EK54 Tnp and a new OE/IE mosaic end sequence containing the OE base-pairs 10, 11 and 12 gives rise to an extraordinarily high transposition frequency. Just as the Tnp is a multifunctional protein, the nucleotides in the 19 bp Tn5 ends also affect other functions besides Tnp binding. Furthermore, the fact that we were able to isolate end sequence variants that transpose at a higher frequency than the natural ends (OE and IE) with wt Tnp reveals yet another way in which the wt transposition frequency is depressed, i.e. by keeping the end sequences suboptimal.

Tn5 in vitro transposition.

This communication reports the development of an efficient in vitro transposition system for Tn5. A key component of this system was the use of hyperactive mutant transposase. The inactivity of wild type transposase is likely to be related to the low frequency of in vivo transposition. The in vitro experiments demonstrate the following: the only required macromolecules for most of the steps in Tn5 transposition are the transposase, the specific 19-bp Tn5 end sequences, and target DNA; transposase may not be able to self-dissociate from product DNAs; Tn5 transposes by a conservative "cut and paste" mechanism; and Tn5 release from the donor backbone involves precise cleavage of both 3' and 5' strands at the ends of the specific end sequences.