Mechanism of Transposition-Explainattion/Animation(3D)
Several different mechanisms of transposition are employed by prokaryotic transposable elements. And, as we shall see later, eukaryotic elements exhibit still additional mechanisms of transposition.
In E. coli, we can identify replicative and conservative (nonreplicative) modes of transposition. In the replicative pathway, a new copy of the transposable element is generated in the transposition event. The results of the transposition are that one copy appears at the new site and one copy remains at the old site. In the conservative pathway, there is no replication. Instead, the element is excised from the chromosome or plasmid and is integrated into the new site.
Replicative transposition
When transposition is from one locus to a second locus for certain transposons, a copy of the transposable element is left behind at the first locus. An analysis of transposon mutants revealed an interesting fact about the mechanism of transposition. Using the transposon Tn3 (Figure 20-22), researchers grouped the mutations that prevent transposition into two categories. A trans-recessive class maps in the gene that encodes the transposase enzyme, a catalyst of transposition. A second class of cis-dominant mutations results in the buildup of an intermediate in the transposition process.Figure 20-23 diagrams the transposition pathway in the Tn3 transposition from one plasmid to another. The intermediate is a double plasmid, with both donor and recipient plasmid being fused together. The combined circle resulting from the fusion of two circular elements is termed acointegrate. Apparently, the mutations in this second class delete a region on the transposon at which a recombination event takes place that resolves cointegrates into two smaller circles. This region, called the internal resolution site (IRS), appears in Figure 20-22.
Figure 20-22
The structure of Tn3. Tn3 contains 4957 base pairs and encodes three polypeptides: the transposase is required for transposition, the repressor is a protein that regulates the transposase gene (see Chapter 11), and β-lactamase confers ampicillin (more...)
Figure 20-23
Transposition of Tn3 takes place through a cointegrate intermediate. Cointegrates in Tn3 transposition are observed for some internal deletions in the transposon. The correct explanation for this observation is that the cointegrates are intermediates (more...)
The finding of a cointegrate structure as an intermediate in transposition helped establish a replicativemode of transposition for certain elements. In Figure 20-23, note how the transposable element is duplicated in the fusion event and how the recombination event that resolves the cointegrate into two smaller circles leaves one copy of the transposable element in each plasmid.
Conservative transposition
Some transposons, such as Tn10, excise from the chromosome and integrate into the target DNA. In these cases, DNA replication of the element does not occur, and the element is lost from the site of the original chromosome. Researchers demonstrated this lack of replication by constructing heteroduplexes of λTn10 derivatives containing the lac region of E. coli. The researchers used DNA from Tn10-lacZ+ and Tn10-lacZ− derivatives. The heteroduplexes, therefore, contain one strand with the wild-type lac region and a second strand with the mutated (Z−) lac region. Figure 20-24 diagrams this part of the experiment. The heteroduplex DNA is used to infect cells that have no lac genes, and transpositions of the TetR Tn10 are selected. Different types of colonies arise from the transpositionof a heteroduplex Z−/Z+ carrying transposon (Figure 20-25). If replication takes place (the replicativemode of transposition), all colonies are either completely Lac+ or completely Lac−, because the replication will convert the heteroduplex DNA into two homoduplex daughter molecules. The mechanism by which this conversion takes place will be examined in detail in the next section. However, if the transposition is conservative and does not include replication, each colony arises from a lacZ+/lacZ− heteroduplex. Such colonies are partly Lac+ and partly Lac−. By using media that stain Lac+ and Lac− cells different colors, researchers can observe the Lac+ and Lac− sectors in colonies.
Figure 20-24
Generation of heteroduplex and homoduplex Tn10 elements. The denaturation and reannealing of a mixture of two parental λ phages carrying Tn10 elements that differ only at three single bases in the transposon yields a mixture of heteroduplex and(more...)
Figure 20-25
Consequences of conservative and replicative transposition. (a) The heteroduplex or homoduplex nature of DNA (see Figure 20-24) is transposed into a target gene. If the starting DNA is heteroduplex, then the resulting DNA will still be heteroduplex only (more...)
Therefore, the determination of whether Tn10 undergoes replicative or conservative transposition can be made by observing whether differently colored sectors exist within the same colony resulting from the transposition. Sectored colonies are observed in a majority of cases (Figure 20-26). Thus, Tn10—and perhaps other transposable elements in E. coli—transpose by excising themselves from the donor DNA and integrating directly into the recipient DNA.
Figure 20-26
Colonies resulting from the experiment illustrated in Figures 20-24 and 20-25. A dye is used that stains Z+ cells blue. One-half of this colony is Z+ (dark area), and the other is Z− (white). (Photograph courtesy of N. Kleckner.)
Molecular consequences of transposition
The molecular consequences of transposition reveal an additional piece of evidence concerning the mechanism of transposition: on integration into a new target site, transposable elements generate a repeated sequence of the target DNA in both replicative and conservative transposition. Figure 20-27depicts the integration of IS1 into a gene. In the example shown, the integration event results in the repetition of a 9-bp target sequence. Analysis of many integration events reveals that the repeated sequence does not result from reciprocal site-specific recombination (as is the case in phage λ integration; see page 229); rather, it is generated in the process of integration itself. The number of base pairs is a characteristic of each element. In bacteria, 9-bp and 5-bp repeats are most common.
Figure 20-27
Duplication of a short sequence of nucleotides in the recipient DNA is associated with the insertion of a transposable element; the two copies bracket the inserted element. Here the duplication that attends the insertion of IS1 is illustrated in a way (more...)
The preceding observations have been incorporated into somewhat complicated models oftransposition. Most models postulate that staggered cleavages are made at the target site and at the ends of the transposable element by a transposase enzyme that is encoded by the element. One end of the transposable element is then attached by a single strand to each protruding end of the staggered cut. Subsequent steps depend on the mode of transposition (replicative or conservative).
Rearrangements mediated by transposable elements
Transposable elements generate a high incidence of deletions in their vicinity. These deletions emanate from one end of the element into the surrounding DNA (Figure 20-28). Such events, as well as element-induced inversions, can be viewed as aberrant transposition events. Transposons also give rise to readily detectable deletions in which part of the element is deleted together with varying lengths of the surrounding DNA. This process of imprecise excision is now recognized as deletions or inversions emanating from the internal ends of the IR segments of the transposon. The process ofprecise excision—the loss of the transposable element and the restoration of the gene that was disrupted by the insertion—also occurs, although at very low rates compared with the frequencies of the events just described.
Figure 20-28
Deletion formation mediated by a transposable element. In this example, the transposable element IS1 is shown at a point in theE. coli chromosome near the gal genes. Deletions can be generated from each end of the IS1 element, extending into the neighboring (more...)
MESSAGE
Some DNA sequences in bacteria and phages act as mobile genetic elements. They are capable of joining different pieces of DNA and are thus capable of splicing DNA fragments into or out of the middle of a DNA molecule. Some naturally occurring mobile or transposable elements carry antibiotic-resistance genes.
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