FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1989701653> ?p ?o ?g. }

• W1989701653 endingPage "10913" @default.
• W1989701653 startingPage "10908" @default.
• W1989701653 abstract "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. 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." @default.
• W1989701653 created "2016-06-24" @default.
• W1989701653 creator A5058864215 @default.
• W1989701653 creator A5075124461 @default.
• W1989701653 date "1998-05-01" @default.
• W1989701653 modified "2023-09-30" @default.
• W1989701653 title "Functional Characterization of the Tn5 Transposase by Limited Proteolysis" @default.
• W1989701653 cites W1539908789 @default.
• W1989701653 cites W1953270925 @default.
• W1989701653 cites W1965127482 @default.
• W1989701653 cites W1966765851 @default.
• W1989701653 cites W1967727152 @default.
• W1989701653 cites W1980829286 @default.
• W1989701653 cites W1991992091 @default.
• W1989701653 cites W2006572311 @default.
• W1989701653 cites W2007605657 @default.
• W1989701653 cites W2009217296 @default.
• W1989701653 cites W2027121633 @default.
• W1989701653 cites W2028816907 @default.
• W1989701653 cites W2030735657 @default.
• W1989701653 cites W2031673350 @default.
• W1989701653 cites W2040512193 @default.
• W1989701653 cites W2064333214 @default.
• W1989701653 cites W2076258014 @default.
• W1989701653 cites W2083729524 @default.
• W1989701653 cites W2086021826 @default.
• W1989701653 cites W2088761341 @default.
• W1989701653 cites W2089882643 @default.
• W1989701653 cites W2104348306 @default.
• W1989701653 cites W2124583402 @default.
• W1989701653 cites W2127558678 @default.
• W1989701653 cites W2132100443 @default.
• W1989701653 doi "https://doi.org/10.1074/jbc.273.18.10908" @default.
• W1989701653 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9556567" @default.
• W1989701653 hasPublicationYear "1998" @default.
• W1989701653 type Work @default.
• W1989701653 sameAs 1989701653 @default.
• W1989701653 citedByCount "20" @default.
• W1989701653 countsByYear W19897016532015 @default.
• W1989701653 crossrefType "journal-article" @default.
• W1989701653 hasAuthorship W1989701653A5058864215 @default.
• W1989701653 hasAuthorship W1989701653A5075124461 @default.
• W1989701653 hasConcept C104317684 @default.
• W1989701653 hasConcept C141231307 @default.
• W1989701653 hasConcept C171250308 @default.
• W1989701653 hasConcept C172510086 @default.
• W1989701653 hasConcept C181199279 @default.
• W1989701653 hasConcept C185592680 @default.
• W1989701653 hasConcept C192562407 @default.
• W1989701653 hasConcept C2780841128 @default.
• W1989701653 hasConcept C2781307694 @default.
• W1989701653 hasConcept C4918238 @default.
• W1989701653 hasConcept C55493867 @default.
• W1989701653 hasConcept C70721500 @default.
• W1989701653 hasConcept C86803240 @default.
• W1989701653 hasConceptScore W1989701653C104317684 @default.
• W1989701653 hasConceptScore W1989701653C141231307 @default.
• W1989701653 hasConceptScore W1989701653C171250308 @default.
• W1989701653 hasConceptScore W1989701653C172510086 @default.
• W1989701653 hasConceptScore W1989701653C181199279 @default.
• W1989701653 hasConceptScore W1989701653C185592680 @default.
• W1989701653 hasConceptScore W1989701653C192562407 @default.
• W1989701653 hasConceptScore W1989701653C2780841128 @default.
• W1989701653 hasConceptScore W1989701653C2781307694 @default.
• W1989701653 hasConceptScore W1989701653C4918238 @default.
• W1989701653 hasConceptScore W1989701653C55493867 @default.
• W1989701653 hasConceptScore W1989701653C70721500 @default.
• W1989701653 hasConceptScore W1989701653C86803240 @default.
• W1989701653 hasIssue "18" @default.
• W1989701653 hasLocation W19897016531 @default.
• W1989701653 hasOpenAccess W1989701653 @default.
• W1989701653 hasPrimaryLocation W19897016531 @default.
• W1989701653 hasRelatedWork W1963486910 @default.
• W1989701653 hasRelatedWork W1989701653 @default.
• W1989701653 hasRelatedWork W2062760434 @default.
• W1989701653 hasRelatedWork W2063654795 @default.
• W1989701653 hasRelatedWork W2414415093 @default.
• W1989701653 hasRelatedWork W2441711217 @default.
• W1989701653 hasRelatedWork W2949911667 @default.
• W1989701653 hasRelatedWork W2952309663 @default.
• W1989701653 hasRelatedWork W2952541249 @default.
• W1989701653 hasRelatedWork W3037456415 @default.
• W1989701653 hasVolume "273" @default.
• W1989701653 isParatext "false" @default.
• W1989701653 isRetracted "false" @default.
• W1989701653 magId "1989701653" @default.
• W1989701653 workType "article" @default.