TATA-Binding Protein Is Limiting for both TATA-Containing and TATA-Lacking RNA Polymerase III Promoters in Drosophila Cells

Abstract

We have investigated the role of the TATA-binding protein (TBP) in modulating RNA polymerase (Pol) III gene activity. Epitope-tagged TBP (e-TBP) was both transiently and stably transfected in Drosophila Schneider S-2 cells to increase the total cellular level of TBP. Analysis of the transcripts synthesized from cotransfected tRNA and U6 RNA genes revealed that both types of RNA Pol III promoters were substantially stimulated by an increase in e-TBP in a dose-dependent manner. Furthermore, a TBP-dependent increase in the levels of endogenous tRNA transcripts was produced in the stable line induced to express the e-TBP. We further determined whether the ability of increased TBP to induce RNA Pol III gene expression was due to a direct effect of increased TBP complexes on RNA Pol III gene promoters or an indirect consequence of enhanced expression of RNA Pol II genes. A TBP expression plasmid (e-TBP332), containing a mutation within the highly conserved carboxy-terminal domain, was both transiently and stably transfected into S-2 cells. e-TBP332 augmented the transcription from two RNA Pol II gene promoters indistinguishably from that observed when e-TBP was expressed. In contrast, e-TBP332 was completely defective in its ability to stimulate either the tRNA or U6 RNA gene promoters. In addition, increasing levels of a truncated TBP protein containing only the carboxy-terminal region failed to induce either the tRNA or U6 RNA gene promoter, whereas it retained its ability to stimulate an RNA Pol II promoter. Thus, the TBP-dependent increase in RNA Pol II gene activity is not sufficient for enhanced RNA Pol III gene transcription; rather, a direct effect on RNA Pol III promoters is required. Furthermore, these results provide the first direct evidence that the amino-terminal region of TBP is important for the formation or function of TBP-containing complexes utilized by TATA-less and TATA-containing RNA Pol III promoters. Together, these studies demonstrate that TBP is limiting for the expression of both classes of RNA Pol III promoters in Drosophila cells and implicate an important role for TBP in regulating RNA Pol III gene expression. RNA polymerase (Pol) III is responsible for the transcription of many small cellular and viral RNAs which are untrans-lated. There are three major classes of promoters that dictate the expression of the genes transcribed by RNA Pol III (43). The tRNA and 5S RNA classes of promoters each contain intragenic promoter elements and generally lack TATA sequences upstream of the gene. Both of these types of promoters require TFIIIB and TFIIIC components to reconstitute transcription in vitro, while the 5S RNA gene additionally uses TFIIIA. The U6 RNA class of promoters require elements that reside exclusively upstream of the gene and include TATA and proximal sequence elements. For the vertebrate systems, this class of promoter requires TFIIIB, TATA-binding protein (TBP), and a distinct multisubunit complex referred to as SNAPc or PTF (18, 29, 44). Although these three genes are representative of the three classes of promoters, there are other RNA Pol III genes that contain features distinct from these classes (for a review, see reference 43) or contain a combination of elements from more than one class of promoters (34). All RNA Pol III promoters appear to require TFIIIB for transcription. In mammalian systems, this component consists of TBP and at least two additional TBP-associated factors (TAFs) (32). One of the human TAF subunits has been identified and cloned (37). Since there is evidence that the SNAPc complex also contains TBP (29), it appears that the TATA-containing U6 RNA promoter uses TBP in a distinct manner compared to the TATA-less tRNA and 5S RNA promoters. TBP has been shown to be necessary for the transcription of all cellular genes. The gene encoding TBP has been cloned from a variety of organisms (19), and the structure of the protein has been resolved by crystallography (3, 27). The car-boxy-terminal domain of approximately 180 amino acids is highly conserved in all organisms and has been shown to contain the DNA binding function (21). Additionally, amino acids that are essential for transcription and are likely involved in the interaction of TBP with other TAFs have been identified in this region (2, 4, 7). TBP is recruited to RNA Pol III TATA-containing promoters through its interaction with DNA, whereas it is recruited to TATA-less promoters via protein-protein interactions with other DNA-bound factors (25, 39). The role of the amino-terminal region of TBP in the formation of RNA Pol I, II, or III transcription initiation complexes is still not clear. This region varies significantly in both length and sequence between different species. Previous studies have used antibodies specific for the amino-terminal region of human TBP to elucidate its role in transcription initiation (23, 26). Studies in yeast have suggested that the amino-terminal region of TBP may be important for transcription of RNA Pol III genes, since human TBP failed to complement a defective yeast TBP and restore RNA Pol III gene expression (6).