Rib pulls down sequences containing known RPG regulator motifs and binds to RPG enhancer DNA in vitro with low affinity and specificity. (A) Top five motifs identified by MEME-ChIP analysis of RPG sequences pulled down with Rib in the SG ChIP-seq. (B) Representation of enriched motifs from A embedded in the 200 nucleotides flanking the TSS of RPGs. The color code for motifs is according to those used in the outlines of A. TSS (+1) were obtained from FlyBase. The top block of enhancers (RpS10B through RpL12) includes genes bound by Rib, and the bottom block (RpL6 through RpS15Ab) includes RPGs not bound by Rib in the ChIP-seq analysis. See Fig. S8 for sequence lineup. (C) T-rich motif for Rib binding from a bacterial one-hybrid analysis with the Rib PSQ domain (source: FlyFactorSurvey; mccb.umassmed.edu/ffs). (D) EMSAs reveal that full-length Rib and its DNA-binding PSQ domain directly bind the RpS17 promoter in a concentration-dependent manner. Left: Rib-dependent mobility shift is indicated by the arrowhead. Right: EMSAs in which the T’s in the RpS17 promoter were replaced with G’s in the TC-rich motif of the RpS17* enhancer. Bottom: CrebA and Rib bind DNA containing the CrebA consensus sites in P24.1 and TSN-2, two bona fide transcriptional targets of CrebA. See Fig. S9 for EMSAs with several additional RPG TSS sequences. All EMSAs were performed twice with identical results. Source data are available for this figure: SourceData F6.