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 transposase in order to assemble an initial domain structure for this protein. The first four amino acids were found to be important for transposition activity but not for DNA binding to the Tn5 outside end (OE). Larger amino-terminal deletions result in the complete loss of transposition in vivo and the concomitant loss of specific DNA binding. Four point 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. Analysis of a series of carboxy-terminal deletions has revealed that the carboxy terminus may actually mask the DNA binding domain, since deletions to residues 388 and 370 result in a large increase in DNA binding activity. In addition, the carboxy-terminal deletion to residue 370 results in a significant increase in the mobility of the Tnp-OE complex indicative of a change in the oligomeric state of this complex. Further carboxy-terminal deletions beyond residue 370 also abolished DNA binding activity. These results indicate that the first four amino acids 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 towards the carboxy terminus, defined by residues 370 to 387, is critical for Tnp multimeric interactions.
Weinreich
M D
MD
Department of Biochemistry, University of Wisconsin, Madison 53706.
Mahnke-Braam
L
L
Reznikoff
W S
WS
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D017354:Point Mutation; D011108:Polymers; D017384:Sequence Deletion; D019895:Transposases
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011108:Polymers; D009713:Nucleotidyltransferases; D019895:Transposases