FIG. 4.

The 8-bp direct repeat binds two molecules of MprA tandemly. (A) Sephacryl-200 gel filtration chromatography was performed on 5 mg (1 mg/ml) of Trx-MprA or 15 mg (3 mg/ml) of several protein standards, including bovine serum albumin (BSA), phosphorylase B (PB), and carbonic anhydrase (CBA). A standard curve (inset) of protein standards was generated to determine the approximate mass of Trx-MprA; ≈76% of Trx-MprA eluted from the column at a molecular mass calculated to be 39.73 kDa. (B) Competitive EMSAs with the direct repeat from the mprA upstream region and recombinant derivatives of MprA; 2.8 ng of a 60-bp PCR-derived DNA probe carrying the direct repeat upstream of mprA was incubated in binding reactions with recombinant forms of MprA. Binding reactions contained no protein (lane 1), 50, 100, or 200 pmol of His-MprA alone (lanes 2 to 4), 200 pmol of His-MprA and 2, 4, 10, 20, 40, 60, 80, 100, 120, or 140 pmol of Trx-MprA (lanes 5 to 14), or 100 pmol of Trx-MprA alone (lane 15). F, free DNA; HH, two molecules of His-MprA; TT, two molecules of Trx-MprA; HT, one molecule each of His-MprA and Trx-MprA.