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Defining functional regions of the IS903 transposase.

Abstract：

:The insertion sequence IS903 encodes a 307 amino acid residue protein, transposase, that is essential for transposition. It is a multi-functional DNA-binding protein that specifically recognizes the 18 bp inverted repeats at the ends of the element and also recognizes DNa non-specifically when it captures a target site. In addition, transposase performs catalytic functions when it mediates the cleavage and religation steps of transposition. We have carried out deletion and mutational analyses to define functional domains of the transposase protein. The deletion studies delineate a 99 residue region of the protein (residues 31 to 129) that specifies binding to the inverted repeat. A slightly larger maltose-binding protein-transposase fusion that includes residues 22 to 139 (Tnp 22-139) binds as efficiently and with the same specificity as the full-length transposase protein. Tnp 22-139 also induces a DNA bend similar to that of the wild-type protein, and so we conclude that all binding and bending specificity is contained within the N-terminal domain of the protein. Unlike full-length transposase, Tnp 22-139 forms additional higher-order complexes in band-shift gels suggesting that the deletion has exposed a surface(s) capable of participating in protein-protein interactions. Six highly conserved residues in the C-terminal portion of the protein were mutated to alanine. Each mutant protein was binding-proficient but defective in transposition. The phenotype of these substitutions, and their alignment with residues shown to abolish catalysis of other transposases and integrases, suggest that these are residues responsible for catalytic steps in transposition of IS903; we believe three of these residues comprise the DDE motif, conserved in transposases and integrases. Our data are consistent with IS903 transposase being composed of two domains: an N-terminal domain primarily involved in DNA binding and a C-terminal domain that is involved in catalysis.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Tavakoli NP,DeVost J,Derbyshire KM

doi

10.1006/jmbi.1997.1410

subject

Has Abstract

pub_date

1997-12-12 00:00:00

pages

491-504

issue

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(97)91410-8

journal_volume

274

pub_type

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Defining functional regions of the IS903 transposase.