BglG and RNAP can be coimmunoprecipitated from cellular extracts. Extracts of cells enriched for [³⁵S]BglG were prepared as described in Material and Methods. The major labeled product was BglG (lane M). The extracts were incubated either without (lane 1) or with (lane 2) antibodies against RNAP. Samples were analyzed by using SDS/PAGE followed by autoradiography. Molecular masses of protein standards are given in kDa.

BglG directly interacts with RNAP. Purified MBP–BglG (lanes 1) or MBP (lanes 2) was incubated with purified RNAP and antibodies against RNAP holoenzyme (A) or against MBP (B), both raised in rabbit. Immunoprecipitated proteins were fractionated by using SDS/PAGE, blotted onto nitrocellulose filters, and probed with antibodies against MBP (A) or against RNAP holoenzyme (B). The filter was reacted with a secondary antibody, goat-anti rabbit, which was detected as described in Material and Methods. The first antibody, which is included in each incubation, also fluoresces up because of the interaction with the secondary antibody. Lane M contains RNAP as a marker.

BglG interacts with the β′ subunit of RNAP. (A) Purified MBP–BglG was incubated with equimolar amounts of purified α (lane 1), β (lane 2), or β′ (lane 3) subunits of RNAP, in the presence of mAbs against the respective subunit. The immunoprecipitated MBP–BglG was detected after SDS/PAGE and Western blot analysis by probing the blot with antibodies against MBP. (B) The individual purified RNAP subunits were fractionated by using SDS/PAGE, blotted onto a nitrocellulose filter, and probed with MBP–BglG, and then with anti-MBP antibodies. (C) Purified MBP–BglG was blotted onto a nitrocellulose filter after SDS/PAGE and probed with the individual RNAP subunits and then with antibodies against the RNAP holoenzyme. (D) Purified α (lane 1), β (lane 2), or β′ (lane 3) were added to MBP–BglG immobilized on amylose resin. After washes, the proteins that eluted with maltose were fractionated by using SDS/PAGE, blotted onto a nitrocellulose filter, and probed with anti-RNAP and anti-MBP antibodies.

BglG interacts with the N-terminal region of β′ subunit. (A) Extracts of cells overproducing β′ (1) or one of the truncated β′ proteins, β′-(1–517 and 1266–1407) (2), β′-(1–876) (3), β′-(545–1407) (4), and β′-(877–1407) (5), were fractionated by using SDS/PAGE, blotted onto a nitrocellulose filter, and probed with MBP–BglG and then with antibodies against MBP (A) or stained with Coomassie blue (B). Arrowheads indicate the positions of β′ and its derivatives. (C) Schematic representation of β′ and its derivatives. Numbers of amino acids are indicated. Boxed areas A to H indicate evolutionarily conserved regions (56). + indicates binding of BglG to the β′ derivative; − indicates lack of binding.