The RNA polymerase II core promoter: a key component in the regulation of gene expression. domain of TBP is a pseudo-symmetric, saddle-shaped molecule with a concave surface that interacts primarily with the TATA element. This binding event induces a sharp bend in the DNA that is thought to be important for the juxtaposition of factors bound both upstream and downstream of the TATA element (21,22). Its convex side is recognized by many transcriptional activators and suppressors (23). The importance of TBP and, in particular, its DNA-binding surface seems to be Rabbit Polyclonal to Histone H2A distinct in different RNA polymerase systems. In an study, different types of transcription showed different sensitivity to TATA-containing DNA oligonucleotides (24), suggesting various roles played by the DNA-binding surface of TBP. Nevertheless, the functions of this surface area of TBP cannot be assessed accurately by using TATA-containing DNA, since RNA polymerase is known to associate with the ends of these DNA oligos non-specifically, thereby causing inhibition of transcription (24). Previously, we isolated and characterized a set of RNA aptamers that bind TBP tightly (25). These aptamers are well-characterized specific molecular probes: they all appear to bind to the concave side of TBP based on their ability to compete with TATA DNA for binding to TBP, yet their modes of interaction with TBP are distinct (25). Here, we describe the utility of these P276-00 aptamers as novel reagents to probe transcription by the three eukaryotic RNA polymerases. The different RNA polymerases responded distinctively to these TBP aptamers. Pol I dependent transcription was completely resistant to all of the TBP aptamers tested. In contrast, Pol II dependent transcription was the most sensitive to TBP aptamers. In crude cell extracts, the aptamers inhibited Pol II dependent transcription even after PICs were formed. Although TBP aptamers inhibited Pol III dependent transcription when they were present during PIC formation, they failed to inhibit transcription after PIC formation. These results revealed that the DNA-binding surface of TBP is involved to different extents in the transcription by different RNA polymerases at both initiation and reinitiation stages. It also revealed a fundamental difference between the stability of the reinitiation intermediate in the Pol P276-00 II system and its counterpart in the Pol III system. The results not only provide insights into the different involvement of TBP in transcription initiation by these RNA polymerases, but they also demonstrate the application of these aptamers for studies of complicated reaction mechanisms as in our analysis of TBP in Pol III transcription. Where aptamers are available, this approach can be generalized to define the role of a particular area on a protein molecule at particular stages of a biological process. MATERIALS AND METHODS P276-00 RNA polymerase I transcription reactions Preparation of whole-cell extract was described previously (25,26). Transcription reactions were carried out essentially according to (26), with minor modifications. The yeast 35S ribosomal gene promoter was used in 20 l reaction mixtures each containing 100 g of yeast whole-cell extract (containing about 20 nM of TBP). The buffer contained 20 mM HEPESCKOH pH 7.9, 50 mM potassium chloride, 10 mM magnesium chloride, 5 mM EGTA, 0.05 mM EDTA, 2.5 mM DTT, 10% glycerol, 100 M each ribonucleoside triphosphate, 10 g/ml -amanitin and template DNA at 10 g/ml (2 nM). The mixture without ribonucleoside triphosphates (NTPs) was incubated at room temperature for 30 min to allow PIC formation. Transcription was started by the addition of NTPs and allowed to proceed for 30 min at room temperature. To inhibit Pol II dependent transcription, 10 g/ml -amanitin was included in the reaction. Reactions were stopped by the addition of 180 l 20 mM.