Purification and biochemical analyses of a monomeric form of Tn5 transposase.

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 (delta369) 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 delta369. 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 time-dependent reaction possibly involving a subunit exchange. Circular permutation assay results indicate that both proteins are capable of distorting the Tn5end sequences upon binding. Molecular weight determinations based on the migratory patterns of complexed DNA in polyacrylamide gels has shown that delta369 specifically binds the Tn5 end sequences as a monomer while full-length Tnp in complex represents a heterodimer.

[1]  J. Calvo,et al.  The Leucine-responsive Regulatory Protein (Lrp) from Escherichia coli , 1996, The Journal of Biological Chemistry.

[2]  W. Reznikoff,et al.  The organization of the outside end of transposon Tn5 , 1996, Journal of bacteriology.

[3]  K. A. Lee,et al.  A monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator , 1994, Molecular and cellular biology.

[4]  W. Reznikoff,et al.  A functional analysis of the Tn5 transposase. Identification of domains required for DNA binding and multimerization. , 1994, Journal of molecular biology.

[5]  W. Reznikoff,et al.  Interaction of Tn5 transposase with the transposon termini. , 1994, Journal of molecular biology.

[6]  W. Reznikoff,et al.  Characterization of the Tn5 transposase and inhibitor proteins: a model for the inhibition of transposition , 1993, Journal of bacteriology.

[7]  J. Mahillon,et al.  The IS4 family of insertion sequences: evidence for a conserved transposase motif , 1993, Molecular microbiology.

[8]  G. May,et al.  An EMSA-based method for determining the molecular weight of a protein--DNA complex. , 1993, Nucleic acids research.

[9]  R. Ebright,et al.  Identification of the activating region of catabolite gene activator protein (CAP): isolation and characterization of mutants of CAP specifically defective in transcription activation. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[10]  G. Maley,et al.  Overcoming inclusion body formation in a high-level expression system. , 1993, Protein expression and purification.

[11]  N. Grindley,et al.  Binding of the IS903 transposase to its inverted repeat in vitro. , 1992, The EMBO journal.

[12]  W. Reznikoff,et al.  Characterization of two hypertransposing Tn5 mutants , 1992, Journal of bacteriology.

[13]  N. Craig,et al.  Interaction of the Tn7-encoded transposition protein TnsB with the ends of the transposon. , 1991, Nucleic acids research.

[14]  E. Getzoff,et al.  DNA‐protein complexes during attachment‐site synapsis in Mu DNA transposition. , 1991, The EMBO journal.

[15]  D. Crothers,et al.  Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[16]  J. F. Thompson,et al.  Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. , 1988, Nucleic acids research.

[17]  W. Reznikoff,et al.  Transcriptional and translational initiation sites of IS50. Control of transposase and inhibitor expression. , 1986, Journal of molecular biology.

[18]  W. Reznikoff,et al.  Control of Tn5 transposition in Escherichia coli is mediated by protein from the right repeat , 1982, Cell.

[19]  S. Halford,et al.  The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies. , 1980, The Biochemical journal.

[20]  W. Reznikoff.  The Tn5 transposon. , 1993, Annual review of microbiology.

[21]  S. McKnight,et al.  Diversity and specificity in transcriptional regulation: the benefits of heterotypic dimerization. , 1991, Trends in biochemical sciences.