Figure 1.

Identification of the host factor that binds specifically to P_mom. (A) P_mom specific activity in crude cell extracts of E. coli. End-labeled 260-bp P_mom fragment (lane 1) was incubated with 220 nM purified C (lane 2) and 1 µg of crude sonicated extract (lane 3) and partially purified cell extract (lane 4) of E. coli MG1655. Lane 5 onwards, PvuI digested P_mom (lane 5) was incubated with 220 nM purified C (lane 6) and 1 µg of crude sonicated extract (lane 7) and partially purified cell extract (lane 8) of E. coli MG1655. EMSAs with cell extracts were carried out in the presence of 200 ng poly (dI – dC). Mobility shift caused by binding of C is depicted by asterisks while that caused by the unknown host factor is depicted by bold arrows. (B) P_mom fragment used for EMSAs. A 260-bp fragment of P_mom obtained upon digestion of plasmid pUW4 with EcoRI and HindIII was radiolabeled at both the 3′-ends (denoted by asterisks) by Klenow polymerase-mediated end filling. Digestion of this fragment with PvuI yields three fragments of sizes 60, 18 and 182 bp of which only the terminal fragments (60 and 182 bp) carry the radiolabel. The larger fragment (182 bp) contains the C-binding site whereas the smaller fragment (60 bp) comes from the upstream region. (C) Effect of ionic strength on stability of the complex. 220-bp P_mom obtained upon digestion of pUW4 with EcoRI and BamHI (lane 1) was incubated with 1 µg of crude cell extract of E. coli MG1655 in the presence of increasing concentrations of NaCl as indicated. The host factor–DNA complex was stable even in 750 mM NaCl. (D) Effect of non-specific competitor DNA on complex formation. End labeled 220 bp P_mom fragment (lane 1) was incubated with 1 µg of crude cell extract of E. coli MG1655 in the presence of 0-, 50- and 100-fold molar excess of three different unlabeled non-specific competitor DNA fragments (lanes 2–10) and unlabeled P_mom fragment (lanes 11–13). Labeled P_mom DNA is competed by the specific unlabeled P_mom DNA but not by any of the three non-specific DNA fragments, showing that the complex results from specific binding of a molecule in the extract to P_mom. (E) LC/ESI-MS analysis of the active fraction. Crude cell extract of E. coli MG1655 was processed as described in ‘Materials and Methods’ section. Eluted fractions from SP Sepharose column that tested positive for P_mom specific DNA binding activity in EMSA were pooled, dialyzed and subjected to LC/ESI–MS for whole protein molecular weight detection. Analysis of the fraction revealed a molecular mass of 11.25 kDa.