title: Involvement of two domains with helix-turn-helix and zinc finger motifs in the binding of IS1 transposase to terminal inverted repeats.
abstract: The insertion element IS1 has two open reading frames (ORFs), insA and insB, and produces a transframe protein InsAB, known as IS1 transposase, by translational frameshifting. The transposase binds to terminal inverted repeats (IRL and IRR) to promote IS1 transposition. Unless frameshifting occurs, IS1 produces InsA protein, which also binds to IRs and therefore acts as an inhibitor of transposition, as well as a transcriptional repressor of the promoter in IRL. A helix-turn-helix (HTH) motif present in both transposase and InsA is thought to be involved in IR-specific DNA binding. A comparison of transposases encoded by IS1 family elements reveals that the N-terminal regions contain four conserved cysteine residues, which appear to constitute a C(2)C(2) zinc finger (ZF) motif. This motif is also thought to be involved in IR-specific DNA binding. In this study, we show that IS1 transposases with an amino acid substitution in the HTH or ZF motif lose the ability to promote transposition. We also show that transposases, as well as InsA proteins with the same substitution, lose the ability to repress the activity of the IRL promoter, and that purified InsA mutant proteins lose the ability to bind to the IRL-containing fragment. Furthermore, we show that InsA protein co-ordinates Zn(II) with the four cysteine residues as ligands and loses the ability to bind to the IRL-containing fragment in the presence of an agent chelating Zn(II). These findings indicate that IS1 transposase has two domains with HTH and ZF motifs responsible for IR-specific DNA binding in promoting transposition. It is assumed that the two domains are needed for transposase to bind to each IR in an oriented manner in order to place a catalytic domain in the C-terminal region of the transposase to a region around the IR end, where the strand transfer reaction occurs in a transpososome.
authors.affiliation: Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Ohta
authors.forename: Etsuro
authors.initials: E
authors.lastname: Yoshimura
authors.forename: Eiichi
authors.initials: E
authors.lastname: Ohtsubo
mesh_terms: D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D019077:Helix-Turn-Helix Motifs / Q000502:physiology*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D016335:Zinc Fingers / Q000502:physiology*
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C088046:InsB protein, E coli; D011506:Proteins; D012097:Repressor Proteins; C064548:insA1 protein, E coli; D004254:DNA Nucleotidyltransferases