Insertion sequence IS2 is a small transposable element which is present in multiple copies in Escherichia coli K-12 and B strains as well as in other enteric bacteria (13,22,28,35). In addition to being a common cause of polar effects (9, 12), IS2 is also responsible for the transposition of the STII enterotoxin gene of enterotoxigenic E. coli (15,23). Nucleotide sequences of IS2 from three different sources have been reported (11,15,34). These three sequences are essentially identical. The entire IS2 is composed of 1,331 bp, which contains a pair of imperfect repeats of 42 bp at the terminus. In the original assignment of nucleotide numbers the IS2 sequence was counted from right to left (11,34). The terminal repeat located on the left-hand end of IS2 was called the right terminal inverted repeat (RIR), and that on the right-hand end was called the left terminal inverted repeat (LIR). These designations have created much confusion and inconvenience. In this report, the coordinates of IS2 are reoriented and the nucleotide positions are renumbered (Fig. 1). For comparisons, the original nucleotide numbers are shown in brackets immediately following the new numbers throughout the text and in the figures.Although the genome of IS2 contains five open reading frames (ORFs) which are capable of encoding proteins greater than 50 amino acids, only one, which has a molecular weight of 14,000, had been detected (14). This protein, InsA (14) A protein of 46 kDa was predicted to be produced via a Ϫ1 frameshift mechanism (7,8,16,30,(36)(37)(38)(39)42). In this communication, we report the detection of this 46-kDa protein of IS2. We also show that this 46-kDa protein can bind both terminal inverted repeats of IS2. MATERIALS AND METHODSRecombinant DNA techniques and materials. Plasmid DNA was isolated by the alkaline lysis method of Birnboim and Doly (3) and purified with a CsClethidium bromide density gradient. Recombinant DNA techniques were performed according to the methods described by Maniatis et al. (26). Restriction enzymes, the E. coli DNA polymerase I Klenow fragment, T4 polynucleotide kinase, and T4 DNA ligase were purchased from Boehringer Mannheim Biochemicals (Indianapolis, Ind.). Chemicals used in this study were obtained from Sigma Chemical Co. (St. Louis, Mo.) or Merck (Darmstadt, Germany).Analysis of IS2-encoded proteins. E. coli JM109(DE3) cells (40) containing pT7insA ( Fig. 2A), pT7insABЈ(A6) (Fig. 2B), or pETinsABЈ(A7) (Fig. 2C) were grown overnight at 37ЊC in 2 ml of M9 medium (26) containing 50 g of ampicillin per ml. This overnight culture was mixed with 2 ml of fresh M9 medium and was further incubated at 37ЊC with shaking until the A 600 of the culture reached 0.4. The cells in the culture were pelleted, washed with M9 buffer (26), and suspended in 1 ml of a methionine assay medium (methionine free) (Difco Laboratories, Detroit, Mich.). After an incubation at 37ЊC for 100 min, isopropyl-␤-D-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 1 mM to induce the production...