A FUNCTIONAL-ANALYSIS OF THE TN5 TRANSPOSASE - IDENTIFICATION OF DOMAINS REQUIRED FOR DNA-BINDING AND MULTIMERIZATION

A series of deletions were constructed in the 476 amino acid Tn5 trans posase in order to assemble an initial domain structure for this prote in. The first four amino acids were found to be important for transpos ition activity but not for DNA binding to the Tn5 outside end (OE). La rger amino-terminal deletions result in the complete loss of transposi tion in vivo and the concomitant loss of specific DNA binding. Four po int mutants and a six base-pair deletion in the amino terminus between residues 20 and 36 were also found to impair DNA binding to the OE. A nalysis of a series of carboxy-terminal deletions has revealed that th e carboxy terminus may actually mask the DNA binding domain, since del etions to residues 388 and 370 result in a large increase in DNA bindi ng activity. In addition, the carboxyterminal deletion to residue 370 results in a significant increase in the mobility of the Tnp-OE comple x indicative of a change in the oligomeric state of this complex. Furt her carboxy-terminal deletions beyond residue 370 also abolished DNA b inding activity. These results indicate that the first four amino acid s of Tnp are important for transposition but not DNA binding, a region between residues 5 and 36 is critical for DNA binding, the wild-type carboxy terminus acts to inhibit DNA binding, and that a region toward s the carboxy terminus, defined by residues 370 to 387, is critical fo r Tnp multimeric interactions.