Figure 2. The PEC contains two ITRs and two transposases. A.

Sequence of the double-strand oligonucleotide (short-PC: short pre-cleaved ITR) used in EMSAs. TS: transferred strand; NTS: non-transferred strand. The 3′ITR is shown in bold, with a 3-bases overhang in 3′ of the TS [14]. Inner sequence: narrow letters. The long-PC oligonucleotide (long pre-cleaved ITR) has the same sequence, with a longer inner sequence. An asterisk marks the position of the ³²P labeling. B. Short/long transposase analyses. EMSAs were performed with 250 nM of short-PC (short pre-cleaved) labeled ITR (as a probe), and 250 nM of purified MBP-MOS1. Lane 1: probe alone, Lane 2: complexes assembly without factor-Xa treatment. Lanes 3 to 5, complexes were subjected to factor-Xa cleavage (1H, 2H and 5H respectively) before electrophoresis. The MOS1 dimer in the PEC was assayed here. We have taken advantage of the fact that the MBP-MOS1 fusion protein contains a cleavage site for factor-Xa between MBP and MOS1. If the PEC contains MBP-MOS1 dimer, then a three-band pattern is expected after cleavage of factor-Xa: one band containing uncleaved MBP-MOS1 (in native PEC, as seen at T = 0), one band containing one cleaved and one uncleaved MOS1 in the complex, and one band containing two cleaved MOS1s in the complex. SEC2 disappears as a result of the factor-Xa cleavage, since it contains two MBP-MOS1 molecules that are converted into MOS1 molecules by the release of the MBP moiety, giving bands with faster mobility in electrophoresis. The proteins present in the various PECs are drawn on the right. C. Short/long ITR analyses. EMSAs were performed with 250 nM of purified MBP-MOS1 and 250 nM of short/long ITR combinations (as indicated). The number of ITRs in the PEC is assayed here. The ITRs present in the complexes are drawn on the right. Short-PC: short pre-cleaved ITR. Long-PC: long pre-cleaved ITR. S*: labeled short-PC. L*: labeled long-PC.