Purification and Biochemical Analyses of a Monomeric Form of Tn5 Transposase

Dona York 0 William S. Reznikoff 0 0 Department of Biochemistry , 420 Henry Mall, University of Wisconsin-Madison , Madison, WI 53706, USA The binding of transposase (Tnp) to the specific Tn5 end sequences is the first dedicated reaction during transposition. In this study, comparative DNA-binding analyses were performed using purified full-length Tnp and a C-terminal deletion variant (D 369) that lacks the putative dimerization domain. The shape of the binding curve of full-length Tnp is sigmoidal in contrast to the hyperbolic-shaped binding curve of D 369. This observation is consistent with previous observations as well as a rate of binding study presented here, which suggest that the full-length Tnp-end interaction, unlike that of the truncated protein, is a complex timedependent reaction possibly involving a subunit exchange. Circular permutation assay results indicate that both proteins are capable of distorting the Tn5 end sequences upon binding. Molecular weight determinations based on the migratory patterns of complexed DNA in polyacrylamide gels has shown that D 369 specifically binds the Tn5 end sequences as a monomer while full-length Tnp in complex represents a heterodimer. - Tn5 is a composite transposon composed of two insertion sequences: IS50R and IS50L that flank antibiotic resistance genes. The functional Tn5 transposase (Tnp) is expressed from IS50R (Fig. 1). The inhibitor protein (Inh), translated from the same reading frame, lacks the N-terminal 55 amino acids. Tnp is a cis-active transposase that specifically recognizes two unique, 19 bp end sequences (OE and IE) positioned at the termini of each IS50. With the exception of position 4, the first 9 bp of each site are identical. The non-identical sections of each site contain binding sites for host proteins (for a review see ref. 1). The critical initial step of Tn5 transposition requires the specific recognition of end sequences by the Tnp protein. Two distinct TnpOE complexes have been observed in gel retardation assays: Complex I and the faster migrating Complex II (2). Depending on the Tnp protein preparations used, wt Tnp or Tnp MA56 (which eliminates production of the Inh protein), the proteins present in Complex I include either Tnp, Inh and a naturally-occurring N-terminal deletion product, Tnp a , or Tnp * To whom correspondence should be addressed and Tnp a , respectively (Fig. 1). Since neither Inh nor Tnp a can themselves form specific nucleoprotein complexes with the OE, their presence in Complex I indicates a proteinprotein interaction with full-length Tnp. The migratory pattern of Complex II, shown to represent binding of C-terminal deletion products, Tnp g and Tnp d (Fig. 1), is indicative of a change in the oligomeric states of these proteins when complexed with the OE (2). The protein composition of Complex I as well as a number of other observations have suggested that the full-length TnpOE interaction is a complicated reaction. Addition of Inh protein stimulates the binding activity of Tnp to the OE presumably through a proteinprotein interaction (3). In addition, a prolonged incubation time increases the amount of TnpOE complex formed (2). This may be related to a disaggregation and subunit exchange between full-length Tnp and Inh resulting in heterooligomer formation. As has been demonstrated for a number of transposases from other systems (46), Tnp also distorts the OE DNA upon binding as determined by a circular permutation assay (7). This distortion is the result of bending as determined by phasing analysis (York and Reznikoff, in preparation). The apparent bending angle is >100 and centers near the first to third nucleotide of the 19 bp OE fragment (7). Preliminary domain mapping of the Tn5 Tnp protein was accomplished by deletion analysis. Various restriction fragments of the Tnp gene were used in an in vitro transcription/translation experiment to generate a family of C-terminal variants of Tnp protein (8). DNA-binding analysis with these Tnp products have suggested that deletion of the C-terminal 107 amino acids restricts Tnp oligomerization. This Tnp variant, D 368, also demonstrated an apparent increased binding affinity. This result implies that the C-terminus of the protein may partially block the DNA-binding domain of the protein, therefore reducing the overall DNA binding affinity for the OE. The goal of this paper is to further characterize the truncated variant of transposase in vitro. A T7 expression vector was made to facilitate the overproduction of a transposase variant lacking 107 amino acids from the C-terminus. The protein was purified using a modified procedure previously reported. Purified preparations of this protein, designated D 369, and full-length Tnp were tested in gel retardation assays for binding affinity and ability to bend the OE. Molecular weight determinations based on the migration patterns of complexed DNA in polyacrylamide gels using both the truncated and full-length protein were also performed which indicated that Complex I represents a heterodimer of Tnp bound to a single OE-DNA fragment while D 369 binds specifically as a monomer. MATERIALS AND METHODS Escherichia coli strains DH5a and BL21(DE3) pLysS were used for plasmid isolation and transposase overexpression, respectively. Plasmids pRZ7074 MA56, pRZ7067 (2), pRZ4826 (8) and pRZ9012 (7) were described previously. pRZ9000 (this study) is described below. Plasmid pRZ9000 used for overexpression of the truncated Tnp, was constructed from pRZ7074 (2) and pRZ4826 (8). pRZ7074 (2), is a pET21d derivative which contains the entire Tnp gene (with MA56 mutation that eliminates Inh production) under control of the IPTG-inducible T7 promoter. Digestion with HindIII results in two fragments: a 6448 bp fragment containing the T7 promoter region and ampicillin resistance gene and the amino-half of the Tnp gene (encoding the first 368 amino acids) and a 1900 bp fragment containing the carboxy-half of the Tnp gene and the rest of the vector. These fragments were treated with Mung Bean nuclease (New England Biolabs) according to manufacturers instructions and the large fragment was isolated and ligated to a 593 bp SmaINruI DNA fragment, isolated from plasmid pRZ4826 (8), which includes stop codons in all three reading frames at the 5 end. The resulting construct, pRZ9000, was confirmed by sequence analysis and used for the overexpression of the truncated form of Tnp [368 amino acids from Tnp and an additional codon (gly) from the stop cassette]. An overnight culture of BL21 (DE3) pLysS containing pRZ9000 MA56 (eliminates production of Inh protein) was used to inoculate 2 l of tryptone-phosphate broth (2% bacto-tryptone, 0.2% Na2HPO4, 0.1% KH2PO4, 0.8% NaCl and 2% yeast extract) (9). Cells were grown at 37 C to an OD600 of 0.5. Protein overexpression was induced with IPTG at a final concentration of 0.1 mM. After an additional 1.5 h incubation, cells were harvested and the protein (...truncated)