Article Title: The autoregulation of a eukaryotic DNA transposon
Figure Lengend Snippet: EMSA analysis of transpososome assembly shows that SEC1 arises from dissociation of the PEC. Transposon ends were encoded on radiolabeled linear DNA fragments. Binding reactions were incubated at 37 °C for 2 hr, separated on a 5% native polyacrylamide gel and recorded by phosphoimaging. ( A ) SEC1 and SEC2 represent a single transposon end bound by a transposase monomer and dimer, respectively (see main text for details). SEC2 comes to dominate the reaction as the transposase concentration rises. There is a significant transition between 4 and 8 nM transposase when SEC1 largely disappears. This corresponds to the point at which the transposon ends become sub-stoichiometric to the transposase dimers. At this point no free transposon ends remain as they are all bound by transposase. According to the S-NEC mechanism (see Figure 1 for details), SEC2 is converted to the transpososome (=PEC) by recruitment of a naked transposon end. OPI occurs when the transposon ends are sub-stoichiometric to transposase dimers and there is a shortage of free transposon ends available for recruitment. Note that the various species observed in these binding reactions are identical to those observed in reactions with the related Mos1 and Himar1 transposons, which display the same behavior for example ( Dawson and Finnegan, 2003 ; Lipkow et al., 2004 ). The present data suggests that the PEC in all three of these related systems is unstable in the EMSA and dissociates into two SEC1 complexes soon after the start of electrophoresis. Thus, in agreement with the S-NEC mechanism, SEC1 disappears at the point in the titration when the transposon ends become sub-stoichiometric to transposase dimers. ( B ) A fixed amount of transposase was titrated with an increasing amount of free transposon ends. The appearance of SEC1 coincides exactly with the appearance of the free transposon ends, which are required for PEC assembly in the S-NEC model. As the amount of transposon ends is increased further, the amount of SEC1 increases. This reflects mass action, which drives PEC assembly by favoring the capture of a free transposon end (see part D below for confirmation). This supports the data in part ( A ) in suggesting that SEC1 arises from the dissociation of the PEC. ( C ) Binding reactions were with a single-chain transposase dimer, in which two monomers are concatenated by a linker peptide joining the C-terminus of one to the N-terminus of the other. Concatenation of the subunits stabilizes the PEC, which is now detected in the gel. As the transposase concentration increases, the PEC disappears at the same point as the free DNA and gives way to SEC2. This behavior is identical to SEC1 in parts ( A ) and ( B ). The single-chain dimer of transposase is fully proficient for the transposition reaction (not shown), demonstrating that SEC1 is not an obligate intermediate of the reaction. This supports the data in parts ( A ) and ( B ) further confirming that SEC1 arises from the dissociation of the PEC. ( D ) In vitro transposition reactions were performed with a plasmid substrate encoding a single transposon end. Reactions were stopped at the indicated times and deproteinated before analysis on a 1.1% agarose TBE gel stained with ethidium bromide. All three sets of transposition reactions contained the same amounts of transposase and supercoiled plasmid substrate. However, the respective reaction volumes were adjusted to 500 µl, 50 µl and 5 µl to achieve the indicated concentrations. Transpososome assembly requires bimolecular synapsis between ends located on different molecules, as illustrated below the gels. This is inefficient owing to the relatively low concentration of one transposon end with respect to another when on separate molecules. When such a transpososome performs cleavage, followed by integration into an unreacted substrate molecule, the product is a linear molecule three times the size of the substrate ( Claeys Bouuaert et al., 2011 ). There is very little reaction when the substrate concentration is low. This reflects the inefficiency of second end recruitment. At high substrate concentration, mass action drives the reaction by favoring second-end recruitment. DOI: http://dx.doi.org/10.7554/eLife.00668.008
Article Snippet: After electrophoresis, the gel was stained with ethidium bromide, destained in water, and photographed on a 310 nm transilluminator using a DC290 camera (Kodak, Rochester, NY) with a 590 DF bandpass filter.
Techniques: Binding Assay, Incubation, Concentration Assay, Electrophoresis, Titration, In Vitro, Plasmid Preparation, Staining