Comparative sequence analysis of IS50/Tn5 transposase

Abstract

Comparative sequence analysis of IS50 transposase-related protein sequences in conjunction with known structural, biochemical, and genetic data was used to determine domains and residues that play key roles in IS50 transposase function. BLAST and ClustalW analyses have been used to find and analyze six complete protein sequences that are related to the IS50 transposase. The protein sequence identity of these six homologs ranged from 25 to 55% in comparison to the IS50 transposase. Homologous motifs were found associated with each of the three catalytic residues. Residues that play roles in transposase-DNA binding, protein autoregulation, and DNA hairpin formation were also found to be conserved in addition to other residues of unknown function. On the other hand, some homologous sequences did not appear to be competent to encode the inhibitor regulatory protein. The results were also used to compare the IS50 transposase with the more distantly related transposase encoded by IS10.

FIG. 1.

IS50 transposition mechanism. IS50 transposition is a multistep process including binding of transposase monomers to the transposon end recognition sequences (so-called cis binding), dimerization of the monomeric transposase-DNA complexes to form the synaptic complex (at this stage trans transposase-DNA contacts are formed), catalysis of the three phosphoryl transfer reactions (3′-end nicking, hairpin formation, and hairpin cleavage), leading to release of the transposon DNA from the donor DNA backbone, target DNA capture, strand transfer of transposon DNA 3′ ends into the target, disengagement of the transposase molecules, and repair of the adjacent single-stranded DNA gaps. This figure is similar to one that was published before (33).

FIG. 2.

A. Clustal W alignment of the IS50 transposase sequence versus six full-length IS50 transposase-like sequences. Active-site residues are white on a black background. Other sequence identities are boxed with * underneath. Residues with a shaded background (and either : or . underneath) denote chemical conservation. Conserved motifs that are discussed further are residues 20 to 31 (IS50 transposase numbering scheme), 38 and 39, 58 to 63, 92 to 103 (DTT), 160 to 166, 183 to 193 (DREAD), 207 to 211, 296 to 306, 319 to 335 (YREK), 342 to 351, 354 to 366, 417 to 425, 429 to 434, and 449 to 452. B. DTT, DREAD, and TYEK active-site motifs.

FIG. 2.

A. Clustal W alignment of the IS50 transposase sequence versus six full-length IS50 transposase-like sequences. Active-site residues are white on a black background. Other sequence identities are boxed with * underneath. Residues with a shaded background (and either : or . underneath) denote chemical conservation. Conserved motifs that are discussed further are residues 20 to 31 (IS50 transposase numbering scheme), 38 and 39, 58 to 63, 92 to 103 (DTT), 160 to 166, 183 to 193 (DREAD), 207 to 211, 296 to 306, 319 to 335 (YREK), 342 to 351, 354 to 366, 417 to 425, 429 to 434, and 449 to 452. B. DTT, DREAD, and TYEK active-site motifs.

FIG. 3.

Molecular location of DTT and DREAD active-site motifs. The locations of the indicated residues are located on the molecular structure of the IS50 transposase bound to the precleaved recognition end DNA sequences. In this and all subsequent figures, the catalytic residues D97, D188, and E326 are shown in yellow space-filled spheres. Conserved motifs are presented in the indicated colors.

FIG. 4.

Conserved N-terminal DNA binding motifs.

FIG. 5.

Conserved C1 region. Residues 296 to 306 (except 298) are in dark blue. W298, which stabilizes the T2 flipped base, is in cyan. The YREK motif (except for the yellow 326) is in purple.

FIG. 6.

Clustal W alignment of IS50 and IS10 C1 regions. The aligned residues are from 298 to 355 (Tn5 transposase numbering scheme). The conservation of the residues is indicated as described for Fig. 2A.