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. IN_HIV-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).

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