Tn5 Transposase

Please note these web pages are part of an assignment for a graduate course in Advanced Biochemistry and Molecular Biology BCMB8010 at the University of Georgia. Questions should be directed to John Buchner

Tn5 Transposase

Abstract

DNA rearrangements are central to the evolutionary process (1). Transposition, first discovered by Barbara McClintock when studying maize genetics, is one of the processes by which organisms rearrange their genomes (2). Transposition is the movement of specific genetic elements from one locus to another (3), and requires three macromolecule elements: the transposon, the transposase, and the target DNA (4). Retroviruses use transposases as a key part of their life cycle (1). Transposition plays a role in horizontal gene transfer in bacteria, including the spread of antibiotic resistance (1). Virulence genes can be regulated by transposition (3). Insertions, deletions, inversions and chromosome fusions can be caused by transposition (4). The Tn5 transposon is a simple composite transposon encompassing drug resistance genes, the IS50 insertion sequences (5), and a crystallized (1, 6) and characterized transposase (1, 4, 7, 8).

The DNA substrate in Tn5 transposition is the IS50 element in the Tn5 transposon (4). The Tn5 transposase targets the outside ends of the IS50 element for binding (5, 9). Mutations and modifications of the target affect binding of the transposase and IS50 DNA strand bending (5, 10). The length of the donor DNA affects transposition (11).

Proteolysis has been used to characterize the different domains of the Tn5 transposase (12). The three domains of Tn5 transposase are an N-terminal domain, the catalytic domain, and the C-terminal domain (1). The N-terminal domain is comprised of a-helices and turns and the primary function is binding DNA (1). The catalytic domain of the Tn5 transposase includes the residues of aspartate97, aspartate188, and glutamate326 (1). This highly conserved DDE motif is the reaction center of most transposases (3). Aspartate97 and glutamate326 coordinate a divalent magnesium cation (1). Tryptophan298 stabilizes a DNA hairpin intermediate of transposition (1). The active site architecture is an a-helix, b-strand, a-helix, and a mixed b-sheet (1). The catalytic region also contains two b-sheets involved in binding of DNA (1). The a-helices of the C-terminal domain are primarily for protein-protein interactions, but do bind DNA (1). Some modifications of the amino acid sequence affect the binding of transposase, the catalytic activity, or the stability of the transposase in a cell (1, 4, 8).

The steps of transposition begin with the Tn5 transposase binding the outside end sequence of DNA (12). This first step is disfavored by sub-optimal interaction of wild type transposase with DNA, sub-optimal sequence of DNA as the target for binding, and inhibition by the C-terminal residues (12). The second step of transposition is synaptic complex formation, and requires the presence of the C-terminal end of the Tn5 transposase (12). The third step in transposition is DNA cleavage initiated by nucleophilic attack followed by DNA hairpin formation (12). This step is followed by target capture and strand transfer, using the free hydroxyl groups of the insertion element to catalyze the integration of the DNA (12). Finally, the transposase is removed (12).

Tn5 transposase can be exploited to make deletions and inversions of genes in vitro (13).

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See the Figures:

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12

Please note that John Buchner created all images and text created for these web pages, unless otherwise noted. The protein structures were created using Rasmol, and files available from the Protein Data Base, including the gif at the top of the page, from file 1F3I.