title: Functional characterization of the Tn5 transposase by limited proteolysis.
abstract: The 476 amino acid Tn5 transposase catalyzes DNA cutting and joining reactions that cleave the Tn5 transposon from donor DNA and integrate it into a target site. Protein-DNA and protein-protein interactions are important for this tranposition process. A truncated transposase variant, the inhibitor, decreases transposition rates via the formation of nonproductive complexes with transposase. Here, the inhibitor and the transposase are shown to have similar secondary and tertiary folding. Using limited proteolysis, the transposase has been examined structurally and functionally. A DNA binding region was localized to the N-terminal 113 amino acids. Generally, the N terminus of transposase is sensitive to proteolysis but can be protected by DNA. Two regions are predicted to contain determinants for protein-protein interactions, encompassing residues 114-314 and 441-476. The dimerization regions appear to be distinct and may have separate functions, one involved in synaptic complex formation and one involved in nonproductive multimerization. Furthermore, predicted catalytic regions are shown to lie between major areas of proteolysis.
authors.affiliation: Department of Biochemistry and the M.D./Ph.D. Program, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Mahnke Braam
authors.forename: W S
authors.initials: WS
authors.lastname: Reznikoff
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002417:Cattle; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D019895:Transposases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019895:Transposases