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PubMed1996

Cis preference of the IS903 transposase is mediated by a combination of transposase instability and inefficient translation.

The transposase protein encoded by the insertion element IS903 belongs to an unusual class of DNA-binding proteins, termed cis-acting proteins, that act preferentially at their site of synthesis. Previous work had led us to propose that instability of the IS903 transposase was a major determinant of its cis preference. Here we describe the isolation of two classes of mutations within the transposase gene that increased action in trans. One class specifically increased trans action without increasing the level of transposition when the mutant gene was located in cis to the transposon. In particular, a threonine-to-proline substitution at amino acid 25 (T25P) reduced cis preference about 60-fold. The half-life of this mutant transposase was significantly longer than that of the wild-type transposase, confirming the critical role of protein instability. The second, larger, class of mutations increased the level of transposition both in trans and in cis. The behaviour and location of these mutations were consistent with an increase in gene expression by improving translational initiation. Several of these mutations exerted a disproportionate effect on the action of transposase in trans, implying that translation efficiency may affect more than just the amount of transposase made. Our results indicate that cis preference of the IS903 transposase is mediated by a combination of transposase instability and inefficient translation initiation.

DNA Nucleotidyltransferases↗

PubMed2001

Comparative architecture of transposase and integrase complexes.

Transposases and retroviral integrases promote the movement of DNA segments to new locations within and between genomes. These recombinases function as multimeric protein-DNA complexes. Recent success in solving the crystal structure of a Tn5 transposase--DNA complex provides the first detailed structural information about a member of the transposase/integrase superfamily in its active, DNA-bound state. Here, we summarize the reactions catalyzed by transposases and integrases and review the Tn5 transposase-DNA co-crystal structure. The insights gained from the Tn5 structure and other available structures are considered together with biochemical and genetic data to discuss features that are likely to prove common to the catalytic complexes used by members of this important protein family.

Binding Sites↗

PubMed2000

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.

Enzyme Activation↗

PubMed2002

A hobo transgene that encodes the P-element transposase in Drosophila melanogaster: autoregulation and cytotype control of transposase activity.

Drosophila were genetically transformed with a hobo transgene that contains a terminally truncated but otherwise complete P element fused to the promoter from the Drosophila hsp70 gene. Insertions of this H(hsp/CP) transgene on either of the major autosomes produced the P transposase in both the male and female germlines, but not in the soma. Heat-shock treatments significantly increased transposase activity in the female germline; in the male germline, these treatments had little effect. The transposase activity of two insertions of the H(hsp/CP) transgene was not significantly greater than their separate activities, and one insertion of this transgene reduced the transposase activity of P(ry(+), Delta2-3)99B, a stable P transgene, in the germline as well as in the soma. These observations suggest that, through alternate splicing, the H(hsp/CP) transgene produces a repressor that feeds back negatively to regulate transposase expression or function in both the somatic and germline tissues. The H(hsp/CP) transgenes are able to induce gonadal dysgenesis when the transposase they encode has P-element targets to attack. However, this ability and the ability to induce P-element excisions are repressed by the P cytotype, a chromosomal/cytoplasmic state that regulates P elements in the germline.

Animals↗

PubMed1987

Construction of hybrid Tn501/Tn21 transposases in vivo: identification of a region of transposase conferring specificity of recognition of the 38-bp terminal inverted repeats.

In order to study the transposase enzymes of Class II prokaryotic transposable elements, we have constructed genes encoding hybrid transposase proteins. This was done by recombination in vivo between the tnpA genes of transposons Tn501 and Tn21. These hybrid genes can complement in trans a transposition-defective mutant of Tn501. The structures of the products of this complementation indicate whether the specificity of the hybrid transposase in recognising the 38 bp terminal inverted repeats is that of Tn501 or that of Tn21. The determinant of this specificity is in the N-terminal region of the transposase protein, between amino acids 28 and 216. The predicted amino acid sequences so far determined of transposases from the Class II family reveal an area of homology in this region.

Amino Acid Sequence↗

PubMed1996

Isolation and characterization of IS10 transposase separation of function mutants: identification of amino acid residues in transposase that are important for active site function and the stability of transposition intermediates.

IS10 transposase mediates end pairing, donor cleavage (or excision) and strand transfer reactions in IS10/Tn10 transposition. Details of how this multi-functional protein mediates these various steps have not been determined. We have used a genetic approach to help define structure-function relationships for IS10 transposase. IS10 transposase mutants that failed to form an excised transposon fragment in vivo and conferred a dominant-negative phenotype were isolated. Mutants with these properties were expected to be defective in catalysis and/or steps preceding donor cleavage. A total of seven mutants were identified, the corresponding proteins were purified and their activities assessed in vitro. Mutant EK292 was found to form wild-type levels of paired end complex, an early intermediate in the reaction, but to be defective in both donor cleavage and strand transfer. This phenotype suggests that E292 comprises part of a catalytic region. Mutants RH119, SF120 and RQ296 formed aberrant paired end complexes, indicating a defect in a step(s) preceding donor cleavage. Mutants WR98, IS101, and AV162 were found to promote donor cleavage in vitro but not strand transfer. For IS101 and AV162 the defect in strand transfer may be related to instability of excision complexes. Finally, the observation that IS101, RH119 and AV162 were greatly suppressed by Mn2+ suggests that these mutations have an effect on Mg2+ interactions with transposase.

Amino Acids↗

PubMed1987

Interactions of the transposase with the ends of Mu: formation of specific nucleoprotein structures and non-cooperative binding of the transposase to its binding sites.

Transposition of the E. coli bacteriophage Mu requires the phage encoded A and B proteins, the host protein HU and the host replication proteins. The ends of the genome of the phage, on which some of these proteins act, both contain three transposase (A) binding sites. The organization of these binding sites on each end, however, is different. Here we show, using DNase footprinting experiments with purified A protein, that mutant A binding sites, which affect transposition, have decreased affinity for the transposase. Furthermore the transposase binds non-cooperatively to all A binding sites both in the left and right end of Mu. Electron microscopic studies show that the A protein forms specific nucleoprotein structures upon binding to the ends of Mu. The A and B proteins interact with the ends of Mu to generate larger structures than with the A protein alone.

Bacteriophage mu↗

PubMed1995

Enhancer-independent variants of phage Mu transposase: enhancer-specific stimulation of catalytic activity by a partner transposase.

Assembly of the functional tetrameric form of phage Mu transposase (A protein) requires specific interactions between the Mu A monomer and its cognate sequences at the ends of the Mu genome (attL and attR) as well as those internal to it (the enhancer element). We describe here deletion variants of Mu A that show enhancer-independence in the assembly of the strand cleavage complex. These deletions remove the amino-terminal region of Mu A required for its interactions with the enhancer elements. The basal enhancer-independent activity of the variant proteins can be stimulated by a partner variant harboring an intact enhancer-binding domain. By exploiting the identical att-binding, and nonidentical enhancer-binding specificities of Mu A and D108 A (transposase of the Mu related phage D108), we show that the stimulation of activity is enhancer-specific. Taken together, these results suggest that the domain of Mu A that includes the enhancer-interacting region may exert negative as well as positive modulatory effects on the strand cleavage reaction. We discuss the implications of these results in the framework of a recent model for the assembly of shared active sites within the Mu A tetramer.

Attachment Sites, Microbiological↗

PubMed1998

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.

Base Sequence↗

PubMed1994

Overexpression of the Tn5 transposase in Escherichia coli results in filamentation, aberrant nucleoid segregation, and cell death: analysis of E. coli and transposase suppressor mutations.

Overexpression of the Tn5 transposase (Tnp) was found to be lethal to Escherichia coli. This killing was not caused by transposition or dependent on the transpositional or DNA binding competence of Tnp. Instead, it was strictly correlated with the presence of a wild-type N terminus. Deletions removing just two N-terminal amino acids of Tnp resulted in partial suppression of this effect, and deletions of Tnp removing 3 or 11 N-terminal amino acids abolished the killing effect. This cytotoxic effect of Tnp overexpression is accompanied by extensive filament formation (i.e., a defect in cell division) and aberrant nucleoid segregation. Four E. coli mutants were isolated which allow survival upon Tnp overexpression, and the mutations are located at four discrete loci. These suppressor mutations map near essential genes involved in cell division and DNA segregation. One of these mutations maps to a 4.5-kb HindIII region containing the ftsYEX (cell division) locus at 76 min. A simple proposition which accounts for all of these observations is that Tnp interacts with an essential E. coli factor affecting cell division and/or chromosome segregation and that overexpression of Tnp titrates this factor below a level required for viability of the cell. Furthermore, the N terminus of Tnp is necessary for this interaction. The possible significance of this phenomenon for the transposition process is discussed.

Cell Division↗

PubMed1997

Examination of the Tn5 transposase overproduction phenotype in Escherichia coli and localization of a suppressor of transposase overproduction killing that is an allele of rpoH.

Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. Tnp overproduction causes cell filamentation, abnormal chromosome segregation, and an increase in anucleated cell formation. There are two simple explanations for the observed phenotype: induction of the SOS response or of the heat shock response. The data presented here show that overproduction of Tnp neither induces an SOS response nor a strong heat shock response. However, our experiments do indicate that induction of some sigma32-programmed function(s) (either due to an rpoH mutation, a deletion of dnaK, or overproduction of sigma32) suppresses Tnp overproduction killing. This effect is not due to overproduction of DnaK, DnaJ, or GroELS. In addition, Tnp but not deltall Tnp (whose overproduction does not kill the host cells) associates with the inner cell membrane, suggesting a possible correlation between cell killing and Tnp membrane association. These observations will be discussed in the context of a model proposing that Tnp overproduction titrates an essential host factor(s) involved in an early cell division step and/or chromosome segregation.

Bacterial Proteins↗

PubMed1999

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.

Bacterial Proteins↗

PubMed2001

DNA-binding activity and subunit interaction of the mariner transposase.

Mos1 is a member of the mariner/Tc1 family of transposable elements originally identified in Drosophila mauritiana. It has 28 bp terminal inverted repeats and like other elements of this type it transposes by a cut and paste mechanism, inserts at TA dinucleotides and codes for a transposase. This is the only protein required for transposition in vitro. We have investigated the DNA binding properties of Mos1 transposase and the role of transposase-transposase interactions in transposition. Purified transposase recognises the terminal inverted repeats of Mos1 due to a DNA-binding domain in the N-terminal 120 amino acids. This requires a putative helix-turn-helix motif between residues 88 and 108. Binding is preferentially to the right hand end, which differs at four positions from the repeat at the left end. Cleavage of Mos1 by transposase is also preferentially at the right hand end. Wild-type transposase monomers interact with each other in a yeast two-hybrid assay and we have used this to isolate mutations resulting in reduced interaction. These mutations lie along the length of the protein, indicating that transposase-transposase interactions are not due to a single interaction domain. One such mutation which retains both DNA-binding and catalytic activity has greatly reduced ability to excise Mos1 from plasmid DNA through coordinate cleavage of the two ends and transposition in vitro is lowered to a level 20-fold below that of the wild-type. This suggests that transposase-transposase interaction is required to form a synaptic complex necessary for coordinate cleavage at the ends of Mos1 during transposition. This mutant enzyme allows insertion at dinucleotides other than TA, including sequences with GC base pairs. This is the first example of a mariner/Tc1 transposase with altered target specificity.

Amino Acid Sequence↗

PubMed2002

Presence of a characteristic D-D-E motif in IS1 transposase.

Transposases encoded by various transposable DNA elements and retroviral integrases belong to a family of proteins with three conserved acidic amino acids, D, D, and E, constituting the D-D-E motif that represents the active center of the proteins. IS1, one of the smallest transposable elements in bacteria, encodes a transposase which has been thought not to belong to the family of proteins with the D-D-E motif. In this study, we found several IS1 family elements that were widely distributed not only in eubacteria but also in archaebacteria. The alignment of the transposase amino acid sequences from these IS1 family elements showed that out of 14 acidic amino acids present in IS1 transposase, three (D, D, and E) were conserved in corresponding positions in the transposases encoded by all the elements. Comparison of the IS1 transposase with other proteins with the D-D-E motif revealed that the polypeptide segments surrounding each of the three acidic amino acids were similar. Furthermore, the deduced secondary structures of the transposases encoded by IS1 family elements were similar to one another and to those of proteins with the D-D-E motif. These results strongly suggest that IS1 transposase has the D-D-E motif and thus belongs to the family of proteins with the D-D-E motif. In fact, mutant IS1 transposases with an amino acid substitution for each of the three acidic amino acids possibly constituting the D-D-E motif were not able to promote transposition of IS1, supporting this hypothesis. The D-D-E motif identified in IS1 transposase differs from those in the other proteins in that the polypeptide segment between the second D and third E in IS1 transposase is the shortest, 24 amino acids in length. Because of this difference, the presence of the D-D-E motif in IS1 transposase has not been discovered for some time.

Amino Acid Motifs↗

PubMed1991

Gamma delta transposase. Purification and analysis of its interaction with a transposon end.

gamma delta, a member of the Tn3 family of prokaryotic transposons, encodes a transposase that binds to the 35-base pair (bp) terminal inverted repeats (IRs) which define the transposing DNA segment. The gamma delta transposase has been overexpressed, identified by molecular weight determination and by immunoblotting, and purified to homogeneity. Production of soluble transposase required the presence of Mg2+ prior to cell lysis. Fractions from a Sephacryl S-300 column contained levels of IR-binding activity that parallel the concentration of transposase, indicating that transposase alone is sufficient for binding to the ends of gamma delta. Hydroxyl radical footprinting indicated that transposase binds to one face of the DNA helix. The protected region extends across the IR and up to 17 bp into the flanking DNA. Integration host factor (IHF), which binds adjacent to transposase, also protects one face of the DNA helix and is shifted about 70 degrees around the helical axis from the transposase protection. Analysis of transposase-DNA complexes by electrophoresis on nondenaturing gels indicated that three complexes, two within the gel and one trapped at the well, result from specific interactions with the IR. The complex in the well and one complex in the gel were analyzed by methylation interference experiments. The results indicate that transposase interacts with specific base pairs between positions 10 and 37 of the IR, a region encompassing three consecutive major and minor grooves. Methylated bases at the very end of the transposon (positions 1-9) and in the flanking DNA did not inhibit transposase binding. Thus, although transposase seems to be in intimate contact throughout the IR of gamma delta and 17 bp of flanking DNA, specific base pair recognition needed for binding appears to be determined by the inner three-quarters of the IR.

Binding Sites↗

PubMed1990

Role of instability in the cis action of the insertion sequence IS903 transposase.

An unusual subset of DNA-binding proteins, termed cis-acting proteins, has been shown to act preferentially at their site of synthesis; the transposases of several bacterial insertion sequences (ISs) fall into this class. The transposase of IS903 exhibits a strong preference for action in cis: complementation of defective transposons in trans occurs at less than 1%. Furthermore, transposition mediated by transposase acting in cis is extremely sensitive to the distance between the 3' end of the transposase gene and the nearest transposon inverted repeat; we find that an insertion of 1 kilobase of DNA reduces transposition to 1-2% of control levels. Here we show that there is a strong correlation between the stability of transposase and its ability to act in trans. We found that the wild-type transposase is a very unstable protein with a physical half-life of about 3 min. However, a transposase-beta-galactosidase fusion protein has a much greater half-life and can act equally well in cis or in trans. In addition, the native transposase is stabilized in lon- strains of Escherichia coli, and, in these protease-deficient strains, trans action of transposase is increased 10- to 100-fold. These results suggest that instability of the IS903 transposase is a major determinant of its cis action and that the La protease, product of the lon gene, is an important determinant of transposase instability.

DNA Transposable Elements↗

PubMed2003

The C-terminus of the Hermes transposase contains a protein multimerization domain.

Transposase activity that mediates the mobility of class II transposable elements, is most commonly initiated by the assembly of higher order synaptic complexes, called transpososomes. The formation of these complexes, that contain the transposable element's DNA as well as two or more molecules of the transposase, is dependent on interactions between transposase molecules. Using the yeast Two-Hybrid system, we were able to identify three regions mediating multimerization of the Hermes transposase, an element used for germline transformation of insects belonging to the hAT family of transposable elements. One region facilitating protein binding of Hermes transposase molecules was found within the first 252 amino acids of the transposase. The second region was located at the C-terminus of the transposase, and was found to be specific for Hermes transposase multimerization. Amino acids 551-569 were not only required for multimerization but were also necessary for transposition of the element. The third region was located between amino acids 253 and 380 and was found to eliminate the non-specific protein binding ability of the N-terminal protein interaction region but was required for the specific protein binding ability of the C-terminal region of the transposase. Five point mutations affecting the structural integrity of the C-terminal multimerization region abolished or significantly reduced transpositional activity. The same region had been previously identified to mediate dimerization in Activator (Ac), another hAT element, indicating that hAT transposase multimerization is likely to be a prerequisite for mobility of their elements.

Amino Acid Sequence↗

PubMed1996

Specific nicking at the 3' ends of the terminal inverted repeat sequences in transposon Tn3 by transposase and an E. coli protein ACP.

BACKGROUND: Tn3, a bacterial transposon, carries tnpA gene encoding transposase which is essential for its transposition. The transposition of Tn3 has been reproduced in vitro in a cell extract containing transposase by using a plasmid carrying mini-Tn3 as the donor and another plasmid as the target. Transposase has the ability to bind to the 38-bp terminal inverted repeats (IRs) of Tn3. The molecular mechanism of the initiation step of the Tn3 transposition reaction promoted by the transposase has, however, not been understood. RESULTS: We found that nicking occurred efficiently in the cell-free system at each of the 3' ends of the IRs of mini-Tn3 in the closed circular or linear donor molecules. The nicking reaction required transposase and Mg2+, but did not require ATP, an ATP-regenerating system, dNTPs and polyvinyl alcohol, which were the requirements for the transposition reaction. By using the nicking assay employed here, transposase was purified almost to homogeneity. Gel filtration and sedimentation analyses indicate that transposase forms a dimer in a solution containing 0.5 M NaCl. The nicking activity of the purified transposase was weak and was found to be stimulated by a host factor. The nicking stimulation factor was subsequently purified and found to be ACP, an Escherichia coli acyl carrier protein. CONCLUSIONS: Nicking occurred efficiently at the 3' ends of mini-Tn3 in the reaction mixture containing transposase and ACP. ACP is known to act as a factor which modulates enzymes that are involved in several biological processes either in the acylated or unacylated form. ACP may also modulate transposase to initiate the transposition reaction with nicking at the 3' ends of Tn3.

Acyl Carrier Protein↗