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Involvement of two domains with helix–turn–helix and zinc finger motifs in the binding of IS 1 transposase to terminal inverted repeats
Author(s) -
Ohta Shinya,
Yoshimura Etsuro,
Ohtsubo Eiichi
Publication year - 2004
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2004.04103.x
Subject(s) - transposase , zinc finger , biology , helix (gastropod) , inverted repeat , terminal (telecommunication) , turn (biochemistry) , genetics , insertion sequence , helix turn helix , zinc , dna binding protein , computational biology , biophysics , transposable element , transcription factor , gene , biochemistry , computer science , telecommunications , ecology , materials science , snail , metallurgy , genome
Summary The insertion element IS 1 has two open reading frames (ORFs), insA and insB , and produces a transframe protein InsAB, known as IS 1 transposase, by translational frameshifting. The transposase binds to terminal inverted repeats (IRL and IRR) to promote IS 1 transposition. Unless frameshifting occurs, IS 1 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 IS 1 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 IS 1 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 IS 1 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.