Figure 5.

 RBP mediates repression at the early stages of preinitiation complex formation. (a) Conditions used to obtain single round transcription with linearized templates in these assays (see Materials and Methods). In all cases, Sp1 and PC4 were added together with the general transcription factors (GTFs). The levels of pIX transcription obtained during preinitiation complex formation are shown as a function of time of addition of RBP repressor (R), or time of addition of TFIIA (IIA) or TFIIH (IIH) in the absence of or presence of RBP repressor as indicated at the top of the lanes. (Vertical arrows) Preincubation times of 30 min before the addition of NTPs. pIX and ML transcripts are indicated on the left. Preinitiation complexes formed in the absence of RBP are protected from repression. Coaddition of RBP during complex formation results in repression. Repression is apparent on later addition of RBP as a function of the presence of TFIIA, but not TFIIH. (b) Conditions of single round transcription as in a but in this case, subcomplexes were preformed for 30 min with specific candidate transcription factors as indicated: TFIID (D), TFIIA (A), and/or TFIIB (B). In all cases, Sp1 and PC4 were added together with the candidate transcription factors. The levels of pIX transcription obtained during preinitiation complex formation are shown as a function of the presence of the specific candidate transcription factors and time of addition of RBP repressor. Arrows at the top of the lanes indicate preincubation times of 30 min before addition of NTPs. pIX and ML transcripts are indicated on the left. Subcomplexes containing TFIID are partially resistant to the later addition of RBP, but are made completely resistant when preformed in the presence of TFIIA, but not TFIIB.