Figure 4.

β Subunit of RNA polymerase makes a direct contact with both iteron-bound λO and a free form of the λO protein. λO (0.5 µM) was bound to oriλ-containing plasmid (2.8 nM) and separated from unbound protein fraction by gel filtration. Subsequently, reaction was supplemented with RNA polymerase (22 nM) and subjected to DSG cross-linking in the presence or absence of nucleotides (500 µM). Free λO protein was also subjected to DSG cross-linking with RNA polymerase. Subsequently, protein complexes were resolved by SDS–PAGE and detected by immunoblotting, performed with polyclonal antibodies specific against λO (A) or monoclonal antibodies against β subunit of RNAP (B) or simultaneously against both proteins (C). The presence or absence of each reaction component is indicated. λO–RNAP polymerase complex was depicted by an arrow.