General features and properties of insertion sequence elements

Previous ...

Co-translational binding and multimerization

Certain prokaryotic IS transposases show a strong preference for acting on the element from which they are expressed rather than on other copies of the same element in the cell. This phenomenon of "cis" preference presumably serves to prevent general activation of several identical IS copies by any "accidental" (stochastic) transposase expression from a single IS. Several different IS such as IS1 (Machida, et al., 1982, Prentki, 1987), IS10 (Morisato, et al., 1983), IS50 (Isberg, et al., 1982), IS903 (Grindley & Joyce, 1981) and IS911 (Duval-Valentin & Chandler, 2011) (see (Nagy & Chandler, 2004) and references therein) exhibit this regulatory phenotype but "cis" preference may be the result of a combination of diverse mechanisms. Thus the Lon protease enhances "cis" preference of the IS903 transposase (Derbyshire, et al., 1990). Transposition is enhanced in the absence of Lon and can be overcome by increased transposase expression (Derbyshire & Grindley, 1996). For IS10, it is influenced by translation levels, Tpase mRNA half-life and translation efficiency (Jain & Kleckner, 1993, Jain & Kleckner, 1993).

Another mechanism, co-translational binding based on tight coupling between prokaryotic transcription and translation, was proposed to explain the inability to complement a Tpase mutant of IS903 (Grindley & Joyce, 1981) and, more specifically, for Tn5 (Sasakawa, et al., 1982).

Some full length IS transposases bind weakly to their cognate IR but the isolated DNA binding domain can bind more strongly. This has been observed for transposases of several elements including IS1 (Zerbib, et al., 1987) and IS30 (Stalder, et al., 1990, Nagy, et al., 2004) and has also been observed for that of IS911. Early studies using band shift assays demonstrated that full length OrfAB binds the IRs only weakly and that OrfA binding was even lower or undetectable (Haren, et al., 2000, Normand, et al., 2001). However, a truncated version of OrfAB, OrfAB[1-149], which is amputated for the C-terminal catalytic domain bound both ends avidly (Haren, et al., 1998) (see also Transposase Stability). It is important to note this implies that, in many in vitro systems, the majority of transposase is thus likely to be inactive or only partially active since it would not bind stably to its substrate. The observations suggest that the C-terminal (C-ter) domain inhibits specific binding by the sequence-specific N-terminal DNA binding domain possibly by steric masking (Fig 1.36.1). This idea is consistent with the observation that IS10 transposase activity is increased by partial denaturation (for example by treatment with low alcohol concentrations; (Chalmers & Kleckner, 1994)). It is also consistent with the observation that the OrfAB protein of IS2 can bind the IS2 IRs when it carries a large GFP tag (Lewis, et al., 2011, Lewis, et al., 2012).

One biological explanation for cis preference is that the nascent N-ter domain might fold before completion of translation of the C-ter domain and the nascent protein could initiate binding directly to the closest IS end. Once bound, it would no longer be sensitive to masking by the C-ter domain. If binding fails to occur after translation of the N-ter DNA binding domain, continuing translation and folding of the C-ter domain would then mask the DNA binding domain resulting in an inactive protein. This implies that binding necessary for subsequent catalysis would occur only transitorily early in translation (Fig 1.36.1).

Direct evidence for co-translational binding was provided for IS911 using an in vitro transcription/translation system (Duval-Valentin & Chandler, 2011) where it was also demonstrated that reducing the efficiency of the -1 translational frameshifting required for IS911 transposase expression resulted in an increase in binding of a nascent transposase peptide. This is presumably because slowing the frameshifting process increases the time that the N-terminal part of the protein (which carries the sequence-specific DNA binding domain) is present on the ribosome enhancing its probability of binding to a neighboring IS end. It is interesting to note that in many IS, the DNA binding domain which recognizes the IR is located at the N-terminal end of the protein which is translated first.

One of the remaining questions concerns transposase multimerization. They must form multimers within the transpososome at some stage in the transposition pathway. Some transposases are monomeric in the absence of DNA (e.g. MuA and Tn5; (Lavoie, et al., 1991), (Braam, et al., 1999, Reznikoff, 2008)) while others are multimeric dimeric in solution ( e.g. INHIV-1 ; (Bao, et al., 2003); (Ren, et al., 2007); (Hare, et al., 2009); (Michel, et al., 2009)).

In view of the possibility that many transposases undergo co-translational binding, and the observation that several different purified full length transposases bind poorly to the ends of their cognate transposon (while the isolated N-terminal DNA binding domain alone binds robustly), it must be emphasized that purified transposases are probably largely inactive. This must be taken into account when assessing transposase properties.

A recent study has provided support for the idea that transposases may also be able to multimerize cotranslationally. This study, has shown that bacterial luciferase subunits LuxA and LuxB may assemble cotranslationally in vivo. This process requires ribosome-associated trigger factor. This chaperone apparently delays subunit interactions until the LuxB dimer interface is available (Shieh, et al., 2015).

    References :
  • Bao KK, Wang H, Miller JK, Erie DA, Skalka AM & Wong I (2003) Functional oligomeric state of avian sarcoma virus integrase. J Biol Chem 278: 1323-1327.
  • Braam LM, Goryshin IY & Reznikoff WS (1999) A Mechanism for Tn5 Inhibition. Carboxyl-terminal dimerization. J.Biol.Chem. 274: 86-92.
  • Chalmers RM & Kleckner N (1994) Tn10/IS10 transposase purification, activation, and in vitro reaction. J Biol Chem 269: 8029-8035.
  • Derbyshire KM & Grindley ND (1996) Cis preference of the IS903 transposase is mediated by a combination of transposase instability and inefficient translation. Mol Microbiol 21: 1261-1272.
  • Derbyshire KM, Kramer M & Grindley ND (1990) Role of instability in the cis action of the insertion sequence IS903 transposase. Proc Natl Acad Sci U S A 87: 4048-4052.
  • Duval-Valentin G & Chandler M (2011) Cotranslational control of DNA transposition: a window of opportunity. Mol Cell 44: 989-996.
  • Grindley ND & Joyce CM (1981) Analysis of the structure and function of the kanamycin- resistance transposon Tn903. Cold Spring Harb.Symp.Quant.Biol. 45 Pt 1: 125-133.
  • Hare S, Di Nunzio F, Labeja A, Wang J, Engelman A & Cherepanov P (2009) Structural basis for functional tetramerization of lentiviral integrase. PLoS Pathog 5: e1000515.
  • Haren L, Polard P, Ton-Hoang B & Chandler M (1998) Multiple oligomerisation domains in the IS911 transposase: a leucine zipper motif is essential for activity. J Mol Biol 283: 29-41.
  • Haren L, Normand C, Polard P, Alazard R & Chandler M (2000) IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif. J Mol Biol 296: 757-768.
  • Isberg RR, Lazaar AL & Syvanen M (1982) Regulation of Tn5 by the right-repeat proteins: control at the level of the transposition reaction? Cell 30: 883-892.
  • Jain C & Kleckner N (1993) IS10 mRNA stability and steady state levels in Escherichia coli: indirect effects of translation and role of rne function. Mol Microbiol 9: 233-247.
  • Jain C & Kleckner N (1993) Preferential cis action of IS10 transposase depends upon its mode of synthesis. Mol Microbiol 9: 249-260.
  • Lavoie BD, Chan BS, Allison RG & Chaconas G (1991) Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome. EMBO J 10: 3051-3059.
  • Lewis LA, Astatke M, Umekubo PT, Alvi S, Saby R & Afrose J (2011) Soluble expression, purification and characterization of the full length IS2 Transposase. Mob DNA 2: 14.

  • Lewis LA, Astatke M, Umekubo PT, et al.(2012) Protein-DNA interactions define the mechanistic aspects of circle formation and insertion reactions in IS2 transposition. Mob DNA 3: 1.

  • Machida Y, Machida C, Ohtsubo H & Ohtsubo E (1982) Factors determining frequency of plasmid cointegration mediated by insertion sequence IS1. Proc Natl Acad Sci U S A 79: 277-281.
  • Michel F, Crucifix C, Granger F, et al. (2009) Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor. Embo J 28: 980-991.
  • Morisato D, Way JC, Kim HJ & Kleckner N (1983) Tn10 transposase acts preferentially on nearby transposon ends in vivo. Cell 32: 799-807.
  • Nagy Z & Chandler M (2004) Regulation of transposition in bacteria. Res Microbiol 155: 387-398.
  • Nagy Z, Szabò M, Chandler M & Olasz F (2004) Analysis of the N-terminal DNA binding domain of the IS30 transposase. Mol Microbiol in press.
  • Normand C, Duval-Valentin G, Haren L & Chandler M (2001) The terminal inverted repeats of IS911: requirements for synaptic complex assembly and activity. J Mol Biol 308: 853-871.
  • Ren G, Gao K, Bushman FD & Yeager M (2007) Single-particle image reconstruction of a tetramer of HIV integrase bound to DNA. J Mol Biol 366: 286-294.
  • Reznikoff WS (2008) Transposon Tn5. Annu Rev Genet 42: 269-286.
  • Sasakawa C, Lowe JB, McDivitt L & Berg DE (1982) Control of transposon Tn5 transposition in Escherichia coli. Proc.Natl.Acad.Sci.U.S.A. 79: 7450-7454.
  • Shieh YW, Minguez P, Bork P, Auburger JJ, Guilbride DL, Kramer G & Bukau B (2015) Operon structure and cotranslational subunit association direct protein assembly in bacteria. Science 350: 678-680.
  • Stalder R, Caspers P, Olasz F & Arber W (1990) The N-terminal domain of the insertion sequence 30 transposase interacts specifically with the terminal inverted repeats of the element. J Biol.Chem. 265: 3757-3762.
  • Zerbib D, Jakowec M, Prentki P, Galas DJ & Chandler M (1987) Expression of proteins essential for IS1 transposition: specific binding of InsA to the ends of IS1. Embo J 6: 3163-3169.