Nucleotide sequences required for Tn3 transposition immunity.

The Tn3 transposon inserts at a reduced frequency into a plasmid already containing a copy of Tn3, a phenomenon known as transposition immunity. The cis-acting site on Tn3 responsible for immunity was mapped by deletions from each side to be within the terminal 38-base-pair sequence that is inversely repeated at the ends of Tn3. Two palindromic sequences are present in the essential part of this region. Some deletions conferred only partial immunity, and others conferred negative immunity. Multiple copies of partially immune ends conferred additional immunity. No other part of Tn3 was necessary for immunity.     

Analysis of Tn3 sequences required for transposition and immunity

Tn3 is a 5-kb transposon (Tn) with 38-bp inverted terminal repeats (ITR). The two 38-bp terminal sequences are required in cis for Tn3 transposition. In this study, the role of the ITR in Tn3 transposition has been further dissected by the use of various mini-Tn3 Tn's. The transposition frequency of these mini-Tn's demonstrate that Tn3 contains no sequence other than the ITR sequences that are necessary for the first step in transposition; the two terminal repeats must be oriented as ITR for transposition to occur; the outside 34 bp of the ITR are required for transposition; and reducing the distance between the terminal sequences does not affect transposition frequency. Moreover, mutant copies of the ITR sequences that cannot function in transposition do not confer transposition immunity.     

Inter- and intramolecular transposition of Tn903.

Summary We have performed a detailed analysis of intra-and intermolecular endproducts of transposition of the compound transposon Tn903 and we show that, in our system, the transposition activity is almost entirely driven by one of the flanking insertion sequences, IS903L. The relatively inactive state of IS903R can be conferred on IS903L by changing the orientation of the internal Tn region. IS903L mediates the formation of the majority of adjacent deletions, insertion/inversions nd cointegrates, all of which are representative of replicative transposition; only a very low level of conservative transposition can be observed. Our results are discussed in relation to those showing that Tn903 uses predominantly the conservative pathway.     

Transposon Tn7. cis-Acting sequences in transposition and transposition immunity

We have identified and characterized the cis-acting sequences at the termini of the bacterial transposon Tn7 that are necessary for its transposition. Tn7 participates in two kinds of transposition event: high-frequency transposition to a specific target site (attTn7) and low-frequency transposition to apparently random target sites. Our analyses suggest that the same sequences at the Tn7 ends are required for both transposition events. These sequences differ in length and nucleotide structure: about 150 base-pairs at the left end (Tn7L) and about 70 base-pairs at the right end (Tn7R) are necessary for efficient transposition. We also show that the ends of Tn7 are functionally distinct: a miniTn7 element containing two Tn7R ends is active in transposition but an element containing two Tn7L ends is not. We also report that the presence of Tn7's cis-acting transposition sequences anywhere in a target replicon inhibits subsequent insertion of another copy of Tn7 into either an attTn7 target site or into random target sites. The inhibition to an attTn7 target site is most pronounced when the Tn7 ends are immediately adjacent to attTn7. We also show that the presence of Tn7R's cis-acting transposition sequences in a target replicon is necessary and sufficient to inhibit subsequent Tn7 insertion into the target replicon.     

Tn3 transposition immunity is conferred by the transposase-binding domain in the terminal inverted-repeat sequence of Tn3

A series of mutant terminal inverted repeats (IRs), having 2 bp substitutions at various sites within the 38-bp IR sequence of the ampicillin-resistance transposon Tn3, were tested for transposition immunity to Tn3. Mutations within region 1-10 in the IR did not affect transposition immunity, while mutations within region 13-38 inactivated the immunity function. These two regions corresponded to domain A which was not bound specifically by Tn3 transposase and to domain B which was bound by the transposase, respectively. This indicates that specific binding of transposase to domain B within the IR sequence is responsible for transposition immunity.     

Absence of cis-acting transposition immunity with Tn7

It is possible in two steps to insert into the plasmid RP4 two copies of the transposon Tn7. This was demonstrated using a wild-type Tn7 in the first step, and a Tn7 derivative (carrying an additional marker), in the second step. The two successive transpositions occurred with the same polarity and frequency. The genetic structures of the resulting plasmids, predicted from the phenotypes of the bacterial host, were confirmed by direct analysis of the plasmid DNAs. Thus, the phenomenon of cis-acting transposition immunity, described with Tn1 or Tn3, does not take place in the case of Tn7.     

Transposition and transposition immunity of transposon Tn3 derivatives having different ends.

Novel Tn1/3 derivatives that contained either two left- or two right-hand ends of the transposon were constructed in a small plasmid. Both transposed at reasonable frequencies to give normal transposition products, suggesting that only the 38-bp inverted repeats of Tn3 are essential for transposition. Plasmids containing transposon derivatives with only one end (either left or right) undergo transposase-dependent transposition between replicons at much lower frequencies, resulting in co-integrate molecules in which there is no substantial duplication of transposon DNA and that appear to be simple fusions of the two plasmids. Both the right and left halves of the transposon are separately able to confer transposition immunity to the plasmid, this immunity being inseparably linked to transposition proficiency and specificity.     

Mutations in the inverted repeats of Tn3 affect binding of transposase and transposition immunity.

In order to better understand the interaction between the inverted repeats (IRs) of the transposon Tn3 and Tn3 transposase, we have looked at the effects of mutations within the IRs on binding of transposase and transposition immunity. Binding of transposase to mutated IRs was measured using a site-specific nitrocellulose filter binding assay and by DNase I protection studies. Transposition immunity was measured in vivo using a transposition mating-out assay. The most important determinants for binding of transposase are present within the inside 21 base-pairs of the IR and several single base-pair mutations significantly reduce binding. Base-pair mutations which do not effect binding have strong negative effects on transposition immunity indicating that simple binding of transposase to the IR is not sufficient for the establishment of transposition immunity.     

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.     

Overproduction of the Tn3 transposition protein and its role in DNA transposition

Five single base pair mutations that increase expression of the tnpA (transposase) gene of the Tn3 transposon approximately 30-fold, but which still allow the gene to be regulated, have been isolated by using a generally applicable procedure that involves distally linked lac gene fusions. The mutations, which are all located in a region controlling initiation of translation of the tnpA gene, do not affect normal repression of tnpA by the tnpR gene product, and yield up to a 9000-fold increase in tnpA protein production when combined with a tnpR mutation and placed on a high copy number plasmid. The mutation yielding the highest expression level was separated from the fused lac gene segment by homologous recombination and was found to increase the rate of transposition without altering the nature of the transposition product; in cells defective in both the E. coli recA gene and the tnpR gene of tn3, cointegrate transposition-intermediate structures occur with the overproducing--as well as with the wild-type--tnpA gene. In the presence of a functional Tn3 tnpR gene or the related transposon delta gamma, such cointegrate structures are resolved into the final products of transposition.     

In vitro transposition of transposon Tn3

Transposition of the ampicillin-resistant transposon Tn3 was reproduced in vitro using the Escherichia coli cell extract. In this cell-free system, we used plasmid DNA carrying mini-Tn3 as donor and phage lambda DNA as target and assayed for ampicillin-resistance transducing phages formed by cointegration of these DNA molecules. Ampicillin-resistance transducing phages, which were obtained by in vitro packaging of lambda DNA after the in vitro transposition reaction, were formed only in the presence of Tn3 transposase. The reaction required mini-Tn3 with the proper sequence and orientation of the terminal inverted repeats of Tn3. The reaction also required DNA synthesis but not RNA synthesis by E. coli RNA polymerase.     

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.     

Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition

The Tn7 transposon avoids inserting into a target DNA that contains a pre-existing copy of Tn7. This phenomenon, known as 'target immunity', is established when TnsB, a Tn7 transposase subunit, binds to Tn7 sequences in the target DNA and mediates displacement of TnsC, a critical transposase activator, from the DNA. Paradoxically, TnsB-TnsC interactions are also required to promote transposon insertion. We have probed Tn7 target immunity by isolating TnsB mutants that mediate more frequent insertions into a potentially immune target DNA because they fail to provoke dissociation of TnsC from the DNA. We show that a single region of TnsB mediates the TnsB-TnsC interaction that underlies both target immunity and transposition, but that TnsA, the other transposase subunit, channels the TnsB-TnsC interaction toward transposition.     

Complementation of transposition of tnpA mutants of Tn3, Tn21, Tn501, and Tn1721

The transposons Tn21, Tn501, and Tn1721 are related to Tn3. Transposition-deficient mutants (tnpA) of these elements were used to test for complementation of transpostion. Transposition of tnpA mutants of Tn501 and Tn1721 was restored by the presence in trans of Tn21, Tn501, and Tn1721, but transposition of a tnpA mutant of Tn21 was restored in trans only by Tn21 itself. Tn3 did not complement transposition of Tn21, Tn501, or Tn1721, and these elements did not complement transposition of Tn3.     

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.     

Polypeptides expressed in Escherichia coli K-12 minicells by transposition elements Tn1 and Tn3.

Escherichia coli K-12 minicells were employed to examine polypeptides encoded by plasmids carrying wild-type and mutant Tn1 or Tn3 transposition elements. Tn1- and Tn3-containing minicells express high levels of four transposon-specified polypeptides. Three, of molecular weights 30,000, 28,000, and 25,000, are related immunologically to beta-lactamase, the enzyme responsible for ampicillin hydrolysis. A fourth polypeptide of molecular weight 19,000 is encoded by the Tn1 or Tn3 region which spans the BamHI cleavage site. Mutant transposons which no longer produce this polypeptide transpose at higher than wild-type frequencies to give aberrant transposition products (Gill et al., J. Bacteriol. 136: 742--756, 1978; Heffron et al., Proc. Natl. Acad. Sci U.S.A. 72:3632--3627, 1975). No expression could be detected from a region of the transposons extending from the inverted repeat sequence distal to the beta-lactamase gene to more than half the distance into the Tn1 or Tn3 sequence.     

Kinetics of Tn5 transposition

The kinetics of Tn5 transposition and gene expression were studied. For about 2 h after infection with lambda Tn5, Tn5 transpositions accumulate, reaching a level of about 1.5% of the infected cells. After 2 h transposition is essentially turned off. In cells carrying a resident Tn5, transposition is undetectable after infection. The synthesis of the Tn5-specific proteins p58 and p54 and the kanamycin-resistance protein were studied in pre-irradiated cells infected with lambda Tn5. The synthesis of p58 and p54 peaked early after infection and was significantly reduced, relative to pneo, by 2 h after infection. Moreover, p54 appeared to reach a maximum later than p58. These kinetic data put new constraints on models for the regulation of Tn5 transposition.