Functional Characterization of the Tn5 Transposase by Limited Proteolysis
1998; Elsevier BV; Volume: 273; Issue: 18 Linguagem: Inglês
10.1074/jbc.273.18.10908
ISSN1083-351X
AutoresLisa A. Mahnke Braam, William S. Reznikoff,
Tópico(s)Chromosomal and Genetic Variations
ResumoThe 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. 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. Transposable DNA elements range in diversity from simple bacterial insertion sequences to complex retroviruses that transpose via RNA intermediates (1Polard P. Chandler M. Mol. Microbiol. 1995; 15: 13-23Crossref PubMed Scopus (189) Google Scholar). Tn5 is a composite transposon found in Gram-negative bacteria and consists of two insertion sequences, IS50R and IS50L, that flank a central region containing antibiotic resistance genes (for a review, see Reznikoff (2Reznikoff W.S. Annu. Rev. Microbiol. 1993; 47: 945-963Crossref PubMed Google Scholar)). IS50R encodes the 476 aa 1The abbreviations used are: aa, amino acid; Tnp, transposase; Inh, transposase inhibitor; OE, transposon outside end; PAGE, polyacrylamide gel electrophoresis; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 1The abbreviations used are: aa, amino acid; Tnp, transposase; Inh, transposase inhibitor; OE, transposon outside end; PAGE, polyacrylamide gel electrophoresis; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. transposase (Tnp) and a nearly identical protein, the transposition inhibitor (Inh). Tnp preferentially acts on Tn5 elements located cisto its site of synthesis, whereas Inh is a trans-acting factor that decreases transposition. The Inh gene is read from the same reading frame as Tnp, but is under the control of separate transcription and translation start sites so that Inh lacks 55 aa of Tnp at the N terminus. In vitro, Tnp is necessary and sufficient for Tn5 transposition in the presence of Mg2+ and specific DNA sequences called outside ends (OE) (3Goryshin I.Y. Reznikoff W.S. J. Biol. Chem. 1998; 273: 7367-7374Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar).Transposition is a multistep process relying on both protein-DNA and protein-protein interactions. In general, the ends of a transposon are bound, synapsed, and cleaved by two or more transposase molecules acting in concert. Subsequently, the protein-transposon complex recognizes target DNA and catalyzes strand transfer, an event which is also called integration. In the case of Tn5, Tnp binds and synapses pairs of OE sites (4de la Cruz N.B. Weinreich M.D. Wiegand T.W. Krebs M.P. Reznikoff W.S. J. Bacteriol. 1993; 175: 6932-6938Crossref PubMed Google Scholar, 5Goryshin I.Y. Kil Y.V. Reznikoff W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10834-10838Crossref PubMed Scopus (32) Google Scholar). Then, Tnp cleaves the transposon free from donor DNA, leaving blunt ends with 3′-hydroxyl groups (3Goryshin I.Y. Reznikoff W.S. J. Biol. Chem. 1998; 273: 7367-7374Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Most likely, each cleavage is the result of two separate nucleophilic attacks on the phosphodiester backbone by water molecules; subsequently, the 3′-hydroxyl groups act as nucleophiles to attack the target DNA and integrate the transposon (6Engelman A. Mizuuchi K. Craigie R. Cell. 1991; 67: 1211-1221Abstract Full Text PDF PubMed Scopus (533) Google Scholar, 7Mizuuchi K. Adzuma K. Cell. 1991; 66: 129-140Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 8van Gent D.C. Mizuuchi D. Gellert M. Science. 1996; 271: 1592-1594Crossref PubMed Scopus (240) Google Scholar). By analogy to the closely related Tn10 Tnp, we hypothesize that these catalytic reactions occur within the same active site with one monomer of a dimeric Tnp unit acting at each OE end (9Bolland S. Kleckner N. Cell. 1996; 84: 223-233Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Inh is known to inhibit Tn5 transposition via nonproductive multimerization with Tnp (4de la Cruz N.B. Weinreich M.D. Wiegand T.W. Krebs M.P. Reznikoff W.S. J. Bacteriol. 1993; 175: 6932-6938Crossref PubMed Google Scholar), and recently, Tnp itself has been shown to inhibit transposition in trans (10Delong A. Syvanen M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6072-6076Crossref PubMed Scopus (17) Google Scholar, 11Wiegand T.W. Reznikoff W.S. J. Bacteriol. 1992; 174: 1229-1239Crossref PubMed Scopus (55) Google Scholar). The cisrestriction of Tnp is also thought to be attributable to nonproductive complex formation (12Weinreich M.D. Gasch A. Reznikoff W.S. Genes Dev. 1994; 8: 2363-2374Crossref PubMed Scopus (54) Google Scholar). Although Inh does not specifically bind DNA, it can form a three-way complex with an OE-bound Tnp monomer (4de la Cruz N.B. Weinreich M.D. Wiegand T.W. Krebs M.P. Reznikoff W.S. J. Bacteriol. 1993; 175: 6932-6938Crossref PubMed Google Scholar, 13York D. Reznikoff W.S. Nucleic Acids Res. 1996; 24: 3790-3796Crossref PubMed Scopus (27) Google Scholar). Inh heterodimerizes with Tnp in solution 2L. A. M. Braam and W. S. Reznikoff, unpublished results. 2L. A. M. Braam and W. S. Reznikoff, unpublished results. and forms homodimers under certain conditions2 (4de la Cruz N.B. Weinreich M.D. Wiegand T.W. Krebs M.P. Reznikoff W.S. J. Bacteriol. 1993; 175: 6932-6938Crossref PubMed Google Scholar).During transposition, different domains and, probably, conformational changes in Tnp are responsible for the following functions: OE binding, synapsis involving Tnp multimerization, catalysis, and target recognition. The N terminus of Tnp is predicted to contain a DNA binding domain (14Zhou M. Reznikoff W.S. J. Mol. Biol. 1997; 271: 362-373Crossref PubMed Scopus (53) Google Scholar, 15Weinreich M.D. Mahnke-Braam L. Reznikoff W.S. J. Mol. Biol. 1994; 241: 166-177Crossref PubMed Scopus (39) Google Scholar). A C-terminal region has been identified as important for protein-protein interactions (15Weinreich M.D. Mahnke-Braam L. Reznikoff W.S. J. Mol. Biol. 1994; 241: 166-177Crossref PubMed Scopus (39) Google Scholar). Tn5 Tnp belongs to the IS4 family of transposases and shares, by sequence alignment with the IS3 and IS15 families and with retroelement integrases, a characteristic transposase/integrase motif probably important for catalysis (16Rezsohazy R. Hallet B. Delcour J. Mahillon J. Mol. Microbiol. 1993; 9: 1283-1295Crossref PubMed Scopus (133) Google Scholar). Here, Tnp is examined by limited proteolysis to further dissect functional aspects of the transposase. Transposable DNA elements range in diversity from simple bacterial insertion sequences to complex retroviruses that transpose via RNA intermediates (1Polard P. Chandler M. Mol. Microbiol. 1995; 15: 13-23Crossref PubMed Scopus (189) Google Scholar). Tn5 is a composite transposon found in Gram-negative bacteria and consists of two insertion sequences, IS50R and IS50L, that flank a central region containing antibiotic resistance genes (for a review, see Reznikoff (2Reznikoff W.S. Annu. Rev. Microbiol. 1993; 47: 945-963Crossref PubMed Google Scholar)). IS50R encodes the 476 aa 1The abbreviations used are: aa, amino acid; Tnp, transposase; Inh, transposase inhibitor; OE, transposon outside end; PAGE, polyacrylamide gel electrophoresis; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 1The abbreviations used are: aa, amino acid; Tnp, transposase; Inh, transposase inhibitor; OE, transposon outside end; PAGE, polyacrylamide gel electrophoresis; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. transposase (Tnp) and a nearly identical protein, the transposition inhibitor (Inh). Tnp preferentially acts on Tn5 elements located cisto its site of synthesis, whereas Inh is a trans-acting factor that decreases transposition. The Inh gene is read from the same reading frame as Tnp, but is under the control of separate transcription and translation start sites so that Inh lacks 55 aa of Tnp at the N terminus. In vitro, Tnp is necessary and sufficient for Tn5 transposition in the presence of Mg2+ and specific DNA sequences called outside ends (OE) (3Goryshin I.Y. Reznikoff W.S. J. Biol. Chem. 1998; 273: 7367-7374Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Transposition is a multistep process relying on both protein-DNA and protein-protein interactions. In general, the ends of a transposon are bound, synapsed, and cleaved by two or more transposase molecules acting in concert. Subsequently, the protein-transposon complex recognizes target DNA and catalyzes strand transfer, an event which is also called integration. In the case of Tn5, Tnp binds and synapses pairs of OE sites (4de la Cruz N.B. Weinreich M.D. Wiegand T.W. Krebs M.P. Reznikoff W.S. J. Bacteriol. 1993; 175: 6932-6938Crossref PubMed Google Scholar, 5Goryshin I.Y. Kil Y.V. Reznikoff W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10834-10838Crossref PubMed Scopus (32) Google Scholar). Then, Tnp cleaves the transposon free from donor DNA, leaving blunt ends with 3′-hydroxyl groups (3Goryshin I.Y. Reznikoff W.S. J. Biol. Chem. 1998; 273: 7367-7374Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Most likely, each cleavage is the result of two separate nucleophilic attacks on the phosphodiester backbone by water molecules; subsequently, the 3′-hydroxyl groups act as nucleophiles to attack the target DNA and integrate the transposon (6Engelman A. Mizuuchi K. Craigie R. Cell. 1991; 67: 1211-1221Abstract Full Text PDF PubMed Scopus (533) Google Scholar, 7Mizuuchi K. Adzuma K. Cell. 1991; 66: 129-140Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 8van Gent D.C. Mizuuchi D. Gellert M. Science. 1996; 271: 1592-1594Crossref PubMed Scopus (240) Google Scholar). By analogy to the closely related Tn10 Tnp, we hypothesize that these catalytic reactions occur within the same active site with one monomer of a dimeric Tnp unit acting at each OE end (9Bolland S. Kleckner N. Cell. 1996; 84: 223-233Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Inh is known to inhibit Tn5 transposition via nonproductive multimerization with Tnp (4de la Cruz N.B. Weinreich M.D. Wiegand T.W. Krebs M.P. Reznikoff W.S. J. Bacteriol. 1993; 175: 6932-6938Crossref PubMed Google Scholar), and recently, Tnp itself has been shown to inhibit transposition in trans (10Delong A. Syvanen M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6072-6076Crossref PubMed Scopus (17) Google Scholar, 11Wiegand T.W. Reznikoff W.S. J. Bacteriol. 1992; 174: 1229-1239Crossref PubMed Scopus (55) Google Scholar). The cisrestriction of Tnp is also thought to be attributable to nonproductive complex formation (12Weinreich M.D. Gasch A. Reznikoff W.S. Genes Dev. 1994; 8: 2363-2374Crossref PubMed Scopus (54) Google Scholar). Although Inh does not specifically bind DNA, it can form a three-way complex with an OE-bound Tnp monomer (4de la Cruz N.B. Weinreich M.D. Wiegand T.W. Krebs M.P. Reznikoff W.S. J. Bacteriol. 1993; 175: 6932-6938Crossref PubMed Google Scholar, 13York D. Reznikoff W.S. Nucleic Acids Res. 1996; 24: 3790-3796Crossref PubMed Scopus (27) Google Scholar). Inh heterodimerizes with Tnp in solution 2L. A. M. Braam and W. S. Reznikoff, unpublished results. 2L. A. M. Braam and W. S. Reznikoff, unpublished results. and forms homodimers under certain conditions2 (4de la Cruz N.B. Weinreich M.D. Wiegand T.W. Krebs M.P. Reznikoff W.S. J. Bacteriol. 1993; 175: 6932-6938Crossref PubMed Google Scholar). During transposition, different domains and, probably, conformational changes in Tnp are responsible for the following functions: OE binding, synapsis involving Tnp multimerization, catalysis, and target recognition. The N terminus of Tnp is predicted to contain a DNA binding domain (14Zhou M. Reznikoff W.S. J. Mol. Biol. 1997; 271: 362-373Crossref PubMed Scopus (53) Google Scholar, 15Weinreich M.D. Mahnke-Braam L. Reznikoff W.S. J. Mol. Biol. 1994; 241: 166-177Crossref PubMed Scopus (39) Google Scholar). A C-terminal region has been identified as important for protein-protein interactions (15Weinreich M.D. Mahnke-Braam L. Reznikoff W.S. J. Mol. Biol. 1994; 241: 166-177Crossref PubMed Scopus (39) Google Scholar). Tn5 Tnp belongs to the IS4 family of transposases and shares, by sequence alignment with the IS3 and IS15 families and with retroelement integrases, a characteristic transposase/integrase motif probably important for catalysis (16Rezsohazy R. Hallet B. Delcour J. Mahillon J. Mol. Microbiol. 1993; 9: 1283-1295Crossref PubMed Scopus (133) Google Scholar). Here, Tnp is examined by limited proteolysis to further dissect functional aspects of the transposase. We thank Dr. Liane Mende-Mueller and the Protein/Nucleic Acids Shared Facility at the Medical College of Wisconsin for peptide sequencing. CD data were obtained, with assistance from Darrell McCaslin, Ph.D., at the University of Wisconsin-Madison Biophysics Instrumentation Facility, which is supported by the University of Wisconsin-Madison and grant BIR-9512577. We thank Terry Arthur for technical assistance with the Far-Western experiment. We also thank Todd Nauman for helpful discussion.
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