IS1 Transposase Research Articles

Analysis of the N-terminal DNA binding domain of the IS30 transposase.

IS30 is the founding member of a large family of widely spread bacterial insertion sequences with closely related transposases. The N-terminal end of the IS30 transposase had been shown to retain sequence-specific DNA binding activity and to protect the IS30 terminal inverted repeats. Structural predictions revealed the presence of a helix-helix-turn-helix motif (H-HTH2) which, in the case of IS30, is preceded by an additional helix-turn-helix motif (HTH1). Analysis of deletion and point mutants in this region revealed that both motifs are important for IS30 transposition. IS30 exhibits two types of insertion specificity preferring either a 24 bp palindromic hot-spot (GOHS) or sequences resembling its ends [left and right terminal inverted repeat (IRL and IRR)]. Results are presented suggesting that the HTH1 region is required for GOHS targeting and interferes with the inverted repeat (IR) targeting. On the other hand, H-HTH2 appears to be required for both. The binding activities of the mutant proteins to the terminal IS30 IRs as measured by gel retardation correlated well with these results. Furthermore, close inspection of the H-HTH2 region revealed significant amino acid identity with a similar predicted secondary structure carried by the transcriptional regulator FixJ of Sinorhizobium meliloti and involved in FixJ binding to its target sequence. This suggests that FixJ and IS30 transposase share similar sequence-specific DNA binding mechanisms.

Functional domains of the IS1 transposase: analysis in vivo and in vitro.

The IS1 bacterial insertion sequence family, considered to be restricted to Enterobacteria, has now been extended to other Eubacteria and to Archaebacteria, reviving interest in its study. To analyse the functional domains of the InsAB' transposase of IS1A, a representative of this family, we used an in vivo system which measures IS1-promoted rescue of a temperature-sensitive pSC101 plasmid by fusion with a pBR322::IS1 derivative. We also describe the partial purification of the IS1 transposase and the development of several in vitro assays for transposase activity. These included a DNA band shift assay, a transposase-mediated cleavage assay and an integration assay. Alignments of IS family members (http://www-is.biotoul.fr) not only confirmed the presence of an N-terminal helix-turn-helix and a C-terminal DDE motif in InsAB', but also revealed a putative N-terminal zinc finger. We have combined the in vitro and in vivo tests to carry out a functional analysis of InsAB' using a series of site-directed InsAB' mutants based on these alignments. The results demonstrate that appropriate mutations in the zinc finger and helix-turn-helix motifs result in loss of binding activity to the ends of IS1 whereas mutations in the DDE domain are affected in subsequent transposition steps but not in end binding.

Involvement of two domains with helix-turn-helix and zinc finger motifs in the binding of IS1 transposase to terminal inverted repeats.

The insertion element IS1 has two open reading frames (ORFs), insA and insB, and produces a transframe protein InsAB, known as IS1 transposase, by translational frameshifting. The transposase binds to terminal inverted repeats (IRL and IRR) to promote IS1 transposition. Unless frameshifting occurs, IS1 produces InsA protein, which also binds to IRs and therefore acts as an inhibitor of transposition, as well as a transcriptional repressor of the promoter in IRL. A helix-turn-helix (HTH) motif present in both transposase and InsA is thought to be involved in IR-specific DNA binding. A comparison of transposases encoded by IS1 family elements reveals that the N-terminal regions contain four conserved cysteine residues, which appear to constitute a C(2)C(2) zinc finger (ZF) motif. This motif is also thought to be involved in IR-specific DNA binding. In this study, we show that IS1 transposases with an amino acid substitution in the HTH or ZF motif lose the ability to promote transposition. We also show that transposases, as well as InsA proteins with the same substitution, lose the ability to repress the activity of the IRL promoter, and that purified InsA mutant proteins lose the ability to bind to the IRL-containing fragment. Furthermore, we show that InsA protein co-ordinates Zn(II) with the four cysteine residues as ligands and loses the ability to bind to the IRL-containing fragment in the presence of an agent chelating Zn(II). These findings indicate that IS1 transposase has two domains with HTH and ZF motifs responsible for IR-specific DNA binding in promoting transposition. It is assumed that the two domains are needed for transposase to bind to each IR in an oriented manner in order to place a catalytic domain in the C-terminal region of the transposase to a region around the IR end, where the strand transfer reaction occurs in a transpososome.

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.

Transposition of IS1 circles.

IS1, the smallest active transposable element in bacteria, encodes transposase. IS1 transposase promotes transposition as well as production of miniplasmids from a plasmid carrying IS1 by deletion of the region adjacent to IS1. The IS1 transposase also promotes production of IS1 circles consisting of the entire IS1 sequence and a sequence, 6-9 bp in length, as a spacer between terminal inverted repeats of IS1. The biological significance of the generation of IS1 circles is not known. Plasmids carrying an IS1 circle with a spacer sequence 6-9 bp long transposed to target plasmids at a very high frequency when transposase was produced from a co-resident plasmid. The products were target plasmids with the donor plasmid inserted at the ends of IS1 in the IS1 circle. This insertion accompanied the removal of the spacer sequence and duplication of the sequence at the target site. IS1 circles with a much longer spacer sequence transposed less frequently. The SOS response was induced in cells harbouring a plasmid with an IS1 circle owing to transposase. IS1 circles could transpose in the strain deficient in H-NS, a nucleoid-associated DNA-binding protein known to be required for the transposition of IS1. IS1 circles appear to act as intermediates for simple insertion into the target DNA via cleavage of the circles which induces the SOS response. H-NS may function in promoting the assembly of an active IS1 DNA-transposase complex at the terminal inverted repeats.

Genetic analyses of the interactions of the IS1-encoded proteins with the left end of IS1 and its insertion hotspot

Insertion sequence IS1 specifies the InsA, ΔInsA-B′-InsB and InsA-B′-InsB protein species. These three proteins have the identical α-helix-turn-α-helix motif that is likely to be responsible for DNA binding. In fact, InsA binds to the ends of IS1, and regulates gene expression and transposition of IS1. ΔInsA-B′-InsB and/or InsA-B′-InsB has been thought to possess a transposase-like activity. Here, I examined the actions of these proteins in vivo on the promoter (pinsL) in the left end of IS1. InsA repressed pinsL-driven gene expression, both in cis and in trans. ΔInsA-B′-InsB inhibited it efficiently only when pinsL was located near the construct where ΔInsA-B′-InsB is expressed. Furthermore, it has been shown that the possible −10 sequence of pinsL is required for ΔInsA-B′-InsB to act on, but the −35 sequence where InsA binds specifically, is not. InsA-B′-InsB appeared not to work on a nearby pinsL. The cis-action of ΔInsA-B′-InsB is consistent with the previous observation that the IS1 transposase acts preferentially in cis. Interestingly, ΔInsA-B′-InsB acted on a nearby P3 promoter in the IS1 insertion hotspot, and on another promoter outside the hotspot. ΔInsA-B′-InsB may generally interact with the regions in or around promoters owing to their low DNA helix stability. Note that IS1 transposes preferentially into A + T-rich DNA segments, and that DNA is unwound from the −10 region of a promoter in transcription. The cis-preference of ΔInsA-B′-InsB would result in an overall reduction of transposition of IS1 and its defective copy in a cell, allowing stable existence of the element in its bacterial host.

IS1-mediated intramolecular rearrangements: formation of excised transposon circles and replicative deletions.

A system is described which permits visualization and analysis of a number of molecular species associated with transposition activity of the bacterial insertion sequence, IS1, in vivo. The technique involves induction of an IS1 transposase gene carried by a plasmid which also includes an IS1-based transposable element. It is, in principle, applicable to the identification of transposition intermediates as well as unstable transposition products and those which are not detectable by genetic means. Thirteen novel molecular species were detected after 4 h of induction. Five major species were characterized, based on their behaviour as a function of time, on their hybridization patterns and on the nucleotide sequences of the transposon-backbone junctions. All result from intramolecular IS1 transposition events. The two reciprocal partner products of IS1-mediated deletions, the intramolecular equivalent of co-integrates generated by intermolecular transposition, have been identified. Both carry a single copy of the transposable element and present complementary distributions of deletion endpoints. These results establish, by direct physical means, that adjacent IS1-mediated deletions are accompanied by duplication of the element. A second type of molecule identified was an excised circular copy of the transposon, raising the possibility that IS1 is capable of following an intermolecular transposition pathway, via excised transposon circles, leading to direct insertion.

Characterization and distribution of insertion sequence IS1239 in Streptococcus pyogenes.

The human pathogenic bacterium Streptococcus pyogenes causes pharyngitis, acute rheumatic fever, glomerulonephritis and toxic-shock-like syndrome. The bacterium synthesizes several extracellular products, including the recently described streptococcal superantigen SSA, a molecule that shares considerable homology with several Staphylococcus aureus enterotoxins. While studying allelic variation at the ssa locus, six isolates expressing serotypes M4, M23, M33, M41, M43, and provisional type PT4854, were identified that had PCR products about 40-bp larger than expected, and one isolate (M15) had an amplified fragment that was more than 1-kb larger than expected. All six isolates have a 34-bp insert located 103 bp 5' of the ssa start codon. The larger product is a result of a 1110-bp insertion at the analogous location. The complementary strand of this insert has a 981-bp open reading frame that potentially encodes a 326-amino-acid polypeptide with substantial homology to the Escherichia coli IS30 transposase. Results of Southern blot analysis showed that at least twelve copies of the sequence are present in the serotype M15 S. pyogenes isolate. This element, designated IS1239, is the first simple insertion sequence described in group-A streptococci. Results of PCR screening showed that 26 of 78 (33%) S. pyogenes isolates expressing distinct M protein serotypes contained sequences with homology to IS1239, which means that the element is widely distributed in the species.

Induction of the SOS Response by IS1 Transposase

We find that IS1 transposase, like that of Tn10, can induce the SOS response when produced at high levels. Most of the activity (> 80%) requires IS1 ends in cis to the transposase gene and depends strictly on the presence of RecBCD function. This implies that processing of transposase-induced cleavages is responsible for generating the response. Induction of the SOS response during growth in a rich medium is seen only when cells approach stationary phase. The end-dependent induction is abolished by mutations in the ends of IS1 that eliminate transposition activity. IS1 ends in identical orientation on the same plasmid are inactive in transposition but stimulate SOS strongly. Even plasmids with a single end can stimulate SOS, probably as a consequence of plasmid dimer formation which places the ends in direct repeat orientation. These results imply that transposase-induced cleavages do not need inversely oriented ends. The system can therefore be used to dissociate cleavage activity from the other reactions of transposition. Induction of SOS by a series of short (67 to 114 bp) IS1-like elements was found to occur in a cyclical pattern as a function of length with a period of 10 to 11 bp. The frequency of cointegration promoted by these elements showed the same helix-phase dependence. These results suggest that transposase molecules bound to the ends of IS1 interact, and that this interaction is needed for the cleavages that initiate transposition.

Is the IS1 transposase, InsAB', the only IS1-encoded protein required for efficient transposition?

The transposase of the bacterial insertion sequence IS1 is normally expressed by inefficient translational frameshifting between an upstream reading frame which itself specifies a transposition inhibitor, InsA, and a second consecutive reading frame located immediately downstream. A fused-frame mutant which carries an additional base pair inserted at the point of frameshifting was constructed. This mutant exhibits high transposition activity and should express the transposase, InsAB', constitutively without frameshifting. Unexpectedly, a second protein species was observed to be expressed from this mutant. We demonstrate here that this protein, InsA*, results from continued frameshifting on the modified frameshift motif. The protein retains the activities of the repressor InsA. Its elimination, by further modification of the frameshift motif, results in a further increase in various transposition activities of IS1. These results support the hypothesis that a single IS1-encoded protein, InsAB', is necessary for transposition.

Characterization of the Caenorhabditis elegans Tc1 transposase in vivo and in vitro.

We have investigated the function of the Tc1A gene of the mobile element Tc1 of Caenorhabditis elegans. Tc1 is a member of a family of transposons found in several animal phyla, such as nematodes, insects, and vertebrates. Two lines of evidence show that Tc1A encodes the transposase of Tc1. First, forced expression of the Tc1A protein in transgenic nematodes results in an enhanced level of transposition of endogenous Tc1 elements. Second, DNase I footprinting and gel retardation assays show that Tc1A binds specifically to the inverted repeats at the ends of the element and that the Tc1A recognition site is located between base pairs 5 and 26 from the ends of Tc1. Functional dissection of the transposase shows the presence of two distinct DNA-binding domains. A site-specific DNA-binding domain is contained within the amino-terminal 63 residues of Tc1A; this region shows sequence similarity to the prokaryotic IS30 transposase. A second, general DNA-binding domain is located between amino acids 71 and 207. Our results suggest that Tc1 is more similar to prokaryotic insertion elements than to eukaryotic transposons such as P elements in Drosophila or Ac and En-1 in plants.

Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements

Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)2 structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA- bacteria, replicated copies of the introduced (IS30)2 structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)2 are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)2, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)2 intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)2 may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)2. Evolutionary implications of these findings are discussed.

The N-terminal domain of the insertion sequence 30 transposase interacts specifically with the terminal inverted repeats of the element.

The gene for the insertion sequence (IS) 30 transposase is placed under the control of the tac promoter, and large quantities of transposase are expressed upon induction. The resulting protein precipitates inside the Escherichia coli cells in the form of inclusion bodies which, upon cell lysis, cannot be dissolved under nondenaturing conditions. In contrast, the N-terminal third of the transposase, a 17-kDa protein produced by a truncated gene, can be purified and is able to interact site specifically with the ends of the IS30 element. In DNase I footprint experiments, regions of 26 nucleotides on one DNA strand and 19 nucleotides on the other strand at either end of the element are protected from nuclease digestion. It is concluded that a functional DNA-binding domain can be formed by expression of only one-third of the complete IS30 transposase. Sequence comparison shows a homology of the IS30 ends to the ends of IS4351 and to the L1 end of bacteriophage Mu.

Frameshifting is required for production of the transposase encoded by insertion sequence 1.

Insertion sequence IS1 has two coding frames, insA and insB, which are essential for its transposition. Here, we show that a frameshifting event in the -1 direction from the 3' end region of the insA frame to an open reading frame (B' frame), extending from the 5' end of the insB frame, is involved in production of the InsA-B'-InsB fusion protein that has IS1 transposase activity. The frameshifting event is likely to have occurred at the sequence AAAAAC where the insA frame overlaps the B' frame. Interestingly, this sequence is also present in one of the two sequences identified in retroviruses as frameshift signals for production of transframe polyproteins from the overlapping genes gag-pro or gag-pro-pol.

A transcriptional terminator sequence in the prokaryotic transposable element IS1.

The prokaryotic transposable element IS1 is known to exert a strong polar effect upon integration into an operon. To elucidate this polar effect, we constructed a plasmid which has an IS1 integrated between the 5' half of the tet gene for tetracycline resistance and the cat structural gene for chloramphenicol resistance. The cat gene is expressed by the tet promoter and the presence of IS1 in orientation I, in which the IS1 transposase genes insA and insB are in the same orientation as the cat gene, reduced the cat expression. By introducing deletions or insertions within the IS1 sequence, we were able to map a rho-dependent terminator TIS1A between the insA and insB genes. Translational interruption between these ins genes is important for TIS1A to be an active terminator.

The phage Mu repressor c and IS30 transposase proteins are significantly related

The IS30 transposase exhibits significant amino acid sequence homology to the phage Mu repressor c in the amino- and carboxy-terminal regions of the proteins. The conserved sequences include the proposed Mu repressor DNA binding site, which is also related to the proposed Mu and D108 transposase DNA binding sites. The carboxy-terminal homologies are characterised by two almost complete, and one partial, somewhat diverged amino acid sequence repeats. Only weak homologies to this domain are present in the Mu transposase (Mu A). Nevertheless, a clear link between an insertion sequence and a bacteriophage has been established.