Escherichia coliInsertion Sequence IS150: Transposition via Circular and Linear Intermediates

Abstract

IS150, a member of the widespread IS3 family, contains two consecutive out-of-phase open reading frames, orfA and orfB, that partially overlap. These open reading frames encode three proteins, InsA, InsB, and the InsAB protein, which is jointly encoded by both open reading frames by means of programmed translational frameshifting. We demonstrate that the InsAB protein represents the IS150 element's transposase. In vivo, the wild-type IS150 element generates circular excision products and linear IS150 molecules. Circular and linear species have previously been detected with mutant derivatives of other members of the IS3 family. Our finding supports the assumption that these products represent true transposition intermediates of members of this family. Analysis of the molecular nature of these two species suggested that the circular forms are precursors of the linear molecules. Elimination of InsA synthesis within the otherwise intact element led to accumulation of large amounts of the linear species, indicating that the primary role of InsA may be to prevent abortive production of the linear species and to couple generation of these species to productive insertion events.

Identification and Characterization of Transposable Elements of Paracoccus pantotrophus

“…A potential frameshift motif (3Ј-AAAAAG-5Ј; position 389) located within a loop of a predicted mRNA stem-loop structure is present within the overlapping region (data not shown). It is probable that this motif (as shown for the IS1 and IS3 family members) (18,26,55) is able to promote a programmed translational frameshifting, which leads to the formation of a functional fusion transposase. Translational slippage in this case might result in the formation of a fusion protein (ORF1 plus ORF2) of 252 aa and a predicted molecular mass of 29.…”

Detection and analysis of transpositionally active head-to-tail dimers in three additional Escherichia coli IS elements

“…The linear form of IS3 was recently shown to be the derivative of the circular one (Sekine et al, 1999). The circular (Welz, 1993) and linear (Haas & Rak, 2002) form of IS150 was also detected, but without any indication of the existence of the dimer. Although IS186 seems not to be related to the other two elements, its activity and the fairly unknown transpositional mechanism give reasons for its investigation.…”

“…Similar to the dimer structures, the covalently joined IRs are also separated by a few base pairs in the circular form of the IS elements. These minicircles can be considered as intermediates of simple insertion, as reported for IS1 (Sekine et al, 1997a;Turlan & Chandler, 1995), IS2 (Lewis & Grindley, 1997), IS3 (Sekine et al, 1994), IS150 (Welz, 1993;Haas & Rak, 2002), IS256 (Prudhomme et al, 2002) and IS911 (Ton-Hoang et al, 1997. The synthetic IS dimer model seems to be applicable for the elements that form both minicircles and dimers (IS2, IS21, IS30, IS256 and IS911); however, its validity might also be extended to additional elements (e.g.…”

“…Such junctions have been identified in IS dimers consisting of two directly repeated elements and in minicircles formed by circularization of a single element. IS dimers have been isolated from IS2 (Szeverényi et al, 1996a), IS21 (Reimmann & Haas, 1987, 1990Reimmann et al, 1989), IS30 (Dalrymple, 1987;Olasz et al, 1993Olasz et al, , 1997, IS256 (Prudhomme et al, 2002) and IS911 (Turlan et al, 2000), and covalently closed circular IS elements have been described for IS1 (Sekine et al, 1997a, Turlan & Chandler, 1995, IS2 (Lewis & Grindley, 1997), IS3 (Sekine et al, 1994), IS30 (Kiss & Olasz, 1999), IS117 (Smokvina et al, 1994), IS150 (Welz, 1993;Haas & Rak, 2002), IS256 (Prudhomme et al, 2002) and IS911 (Polard et al, 1992(Polard et al, , 1996. For some of these elements both circular and dimeric forms have been reported, suggesting a possible connection between the formation and transposition of these structures, as described for IS30 (Kiss & Olasz, 1999).…”

Formation of an inverted repeat junction in the transposition of insertion sequence ISLC3 isolated from Lactobacillus casei

“…The IS3 family elements are produced by transposition in a simple cut-andpaste manner involving several steps. In the first step of transposition, the transposase OrfAB cleaves a single DNA strand at one IS end to generate a free 39-OH group and then transfers this end to an open end of the same strand which is several nucleotides distal from the other IS end (Haas & Rak, 2002;Sekine et al, 1999). A 'figure of eight' is formed by the IS sequence following repair by the host repair machinery and replication by the IS itself at the junction region (Turlan & Chandler, 1995).…”