Fig. 4.

Mapping interacting subdomains of NRPD1 and RDR2. (A) Testing NRPD1 amino acid intervals for interaction with RDR2. Six overlapping polypeptides that collectively represent the 1,453 amino acids of full-length NRPD1 were designed, each having a C-terminal FLAG tag. The polypeptides were then expressed in vitro using a cell-free transcription-translation system (SI Appendix, Fig. S1A) and incubated with V5-tagged recombinant RDR2. Anti-V5 immunoprecipitation was then used to pull down RDR2 and any associated proteins, as depicted in the cartoon. IPed proteins were resolved by SDS-PAGE and subjected to immunoblotting using anti-FLAG and anti-V5 antibodies. Immunoblots were probed with anti-FLAG and anti-V5. The control is a mock reaction in which the bacterial transcription-translation system was not provided with an NRPD1 coding sequence to transcribe or translate prior to incubation with v5-tagged RDR2. (B) NRPD1_1–300 and RDR2 co-IP. Recombinant, FLAG-tagged NRPD1_1–300 expressed in E. coli (SI Appendix, Fig. S1B) was incubated with recombinant V5-tagged RDR2 (lanes 3 and 6), then IPed using either anti-FLAG or anti-V5 resin. In lanes 1, 2, 4, and 5, the individual proteins were IPed as controls. (C) Testing RDR2 amino acid intervals for interaction with NRPD1_1–300. Seven overlapping polypeptides of ∼22 kDa that collectively represent the amino acid sequence of full-length RDR2 (1,133 amino acids) were designed, each having a C-terminal HA epitope tag. The recombinant RDR2 polypeptides were then expressed in vitro using a cell-free transcription-translation system (SI Appendix, Fig. S2A). The RDR2 polypeptides were then incubated with NRPD1_1–300 followed by anti-FLAG IP to pull down NRPD1_1–300 and any associated proteins, as depicted in the cartoon at Top. IPed proteins were resolved by SDS-PAGE and subjected to immunoblotting using anti-FLAG and anti-HA antibodies. Immunoblots were probed with anti-HA and anti-FLAG. The diagram at Bottom shows that RDR2_771–970 includes the enzyme’s active site. The control is a mock reaction in which the bacterial transcription-translation system was not provided with an RDR2 coding sequence to transcribe or translate prior to incubation with FLAG-tagged NRPD1. (D) Recombinant NRPD1_1–300 and recombinant RDR2_771–970, each expressed in E. coli (SI Appendix, Figs. S1B and S2B) were incubated together (lanes 3 and 6) or alone (lanes 1, 2, 4, and 5), then subjected to anti-HA or anti-FLAG IP. IPed proteins were resolved by SDS-PAGE and subjected to immunoblotting using anti-FLAG or anti-HA antibodies.