Figure 5.

RNAi of coactivators reveals distinct roles for both TFIID and MED. (A) S2 cells were treated with dsRNA directed against the TFIID subunit indicated below each lane. Subsequently they were treated with copper for 6 h. Twenty micrograms of total RNA was subjected to primer extension analysis with a primer that anneals to the endogenous MtnA transcript; the arrowhead marks the major start site of transcription. (B) The MtnA RNA level was normalized to the amount of endogenous Rp49 transcript. The data are plotted as the fraction of the level in the non-RNAi-treated cells. The TFIID subunit targeted by dsRNA is indicated below each bar. (C) Protein immunoblot analysis of nuclear extract from S2 cells treated with dsRNA. The RNAi target is listed across the top of the panel. Twenty micrograms of nuclear extract was resolved by SDS-PAGE and transferred to nitrocellulose, and proteins were detected with antibodies directed against the TFIID subunits listed on the left. The MED17 RNAi-treated cells serve as a control for the specificity of the RNAi. (D) Reverse transcriptase PCR analysis of the RNA levels of the TFIID genes. The gene targeted by RNAi is listed across the top of the panel. The genes listed on the left were amplified with primers specific for their RNA. (E) S2 cells were treated with dsRNA directed against the MED subunit indicated below each lane. Subsequently they were treated with copper for 6 h. Twenty micrograms of total RNA was subjected to primer extension analysis with a primer that anneals to the endogenous MtnA transcript; the arrowhead marks the major start site of transcription. (F) The MtnA, MtnB, and MtnD RNA level was normalized to the amount of endogenous Rp49 transcript. The data are plotted as the fraction of the level in the non-RNAi-treated cells. The MED subunit targeted by dsRNA is indicated below each bar. (G) Transient transfection of the MtnA reporter in dsRNA-treated cells. HSF serves as a nonspecific control. The data are plotted as a ratio of MtnA promoter activity to Actin 5C promoter activity.