In the nucleus of eukaryotes, transcription is carried out by three different RNA polymerases, RNA polymerase I, II and III (Pol I, II and III) (1
). Each of them is dedicated to the transcription of distinct sets of genes, but none is able to recognize its target promoters independently. Instead, general transcription factors (GTFs) recognize the promoter elements and recruit the correct RNA polymerase. The TATA-binding protein (TBP) is an essential GTF involved in transcription by all three eukaryotic RNA polymerases (2
). TBP was first identified as a subunit of TFIID, a complex involved in transcription by Pol II (4
), and recognizes an important eukaryotic core promoter motif, the TATA element (8
). TBP is also present in two other complexes that are involved in Pol II dependent transcription, the B-TFIID complex and the TAC complex (9
). Moreover, TBP is a component of complexes required for Pol I (SL1 complex) and Pol III (TFIIIB complex) dependent transcription (10
Different RNA polymerases use diverse mechanisms to control and regulate transcription initiation and reinitiation. Pol I is reserved for the transcription of the large ribosomal RNA genes. To initiate Pol I dependent transcription, the polymerase interacts with promoters complexed with UBF and SL1. TBP and the Pol I-specific TBP-associated factors (TAFs) recruit Pol I directly to the promoter and remain bound to the DNA to support multiple rounds of transcription (14
). A high density of Pol I molecules are loaded onto the rRNA gene when the rDNA template is being transcribed. Polymerases that read through from the upstream rDNA repeat can (in some cases) bypass the requirement for preinitiation complex (PIC) formation (15
). These features contribute to the high transcription efficiency by Pol I.
Pol II is responsible for the transcription of the protein-coding genes and some small nuclear RNAs. Pol II depends on the basal transcription factors TFIID, IIB, IIF, IIE and IIH, and additional upstream activators for accurate and efficient initiation at different promoters. After Pol II leaves the promoter, TFIIB and TFIIF are released, whereas other factors such as activators, TBP, Mediator, TFIIH and TFIIE remain largely promoter-associated and form what is termed a reinitiation intermediate or scaffold, to facilitate subsequent rounds of transcription (16
Pol III transcribes some structural and catalytic RNAs, including most small nuclear RNAs, tRNAs and 5S rRNA (17
). It requires TFIIIB and IIIC for most of its promoters, and in addition, TFIIIA is essential for recognition of the 5S gene promoter (18
). Transcription of Pol III genes also begins with the step-wise assembly of a PIC. When bound to the upstream region of 5S and tRNA genes, yeast TFIIIB correctly positions Pol III with respect to the promoter and supports multiple rounds of transcription (19
). Reinitiation has a higher efficiency than does de novo
initiation in Pol III dependent transcription (20
). The stable association of TFIIIB with promoter, even after Pol III progresses into elongation, bypasses the need for PIC formation, thus accelerating the process of reinitiation.
TBP is a relatively small molecule with a divergent N-terminal domain and a highly conserved C-terminal domain. The C-terminal core 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
). 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 distinct in different RNA polymerase systems. In an in vitro
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 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 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.