Interplay of TBP Inhibitors in Global Transcriptional Control

The TATA-binding protein (TBP) plays a central role in assembling eukaryotic transcription complexes and is subjected to extensive regulation including auto-inhibition of its DNA binding activity through dimerization. Previously, we have shown that mutations that disrupt TBP dimers in vitro have three detectable phenotypes in vivo, including decreased steady-state levels of the mutants, transcriptional derepression, and toxicity toward cell growth. In an effort to more precisely define the multimeric structure of TBP in vivo, the crystallographic dimer structure was used to design mutations that might enhance dimer stability. These mutations were found to enhance dimer stability in vitro and significantly suppress in vivo phenotypes arising from a dimer-destabilizing mutation. Although it is conceivable that phenotypes associated with dimer-destabilizing mutants could arise through defective interactions with other cellular factors, intragenic suppression of these phenotypes by mutations designed to stabilize dimers provides compelling evidence for a crystallographic dimer configuration in vivo.Gene expression levels are derived from a net output of a dynamic interplay of positive and negative regulatory factors. The main steps leading toward gene activation include chromatin modification and remodeling, TBP 1 delivery, and assembly of the RNA polymerase II holoenzyme (1-3). Each of these steps is the target of multiple regulatory factors. TBP delivery to promoters is directed by the positive action of transcriptional activators, acetylated histone tails, SAGA and TFIID delivery complexes, basal factors such as TFIIA and TFIIB, and the TATA box. Counteracting this delivery are Mot1, NC2, a portion of TAF1 (termed TAND), the amino-terminal domain of TBP, and TBP auto-inhibition, which occurs through dimerization and occlusion of its DNA binding surface.TBP auto-inhibition through dimerization is an evolutionary conserved process, occurring from yeast to mammals (4 -15). The structure of TBP dimers has been defined crystallographically and through biochemical analysis. Dimer instability caused by mutations along the crystallographic dimer interface correlate with transcriptional derepression in yeast cells (8,15), and this derepression occurs genome-wide at about 7% of all genes (16). Together, these findings indicated that TBP dimerization represents a physiologically important mechanism for auto-inhibiting its DNA binding activity. Nonetheless, the notion of an autoinhibited TBP dimer has been sufficiently controversial (17) that we pursued the possibility of using the x-ray crystal structure of TBP dimers to design stabilizing mutations that might intragenically suppress a dimerization mutant.We focused on two residues, Arg-98 and Arg-171, which lie on opposite sides of a TBP monomer. In the dimer configuration, Arg-98 of one monomer lies immediately across Arg-171 of the opposing monomer (11). We reasoned that changing one or the other to an acidic residue might generate a positive electrostatic interaction wh...

mentioning

confidence: 88%

Section: Fig 6 Suppression Of Toxicity Associated With Tbp(n69r)mentioning

confidence: 99%

Engineering Dimer-stabilizing Mutations in the TATA-binding Protein

Kou

Pugh

2004

“…Oxygen deficiency induces binding of NC2 to certain mammalian genes correlated to repression of transcription, which argues for a similar role in vivo (37). However, in Saccharomyces cerevisiae, NC2 seems to affect gene transcription both positively and negatively (38,39). Chromatin immunoprecipitation (ChIP) data suggested that the cofactor localizes to both inactive and active genes.…”

mentioning

confidence: 99%

Efficient Binding of NC2·TATA-binding Protein to DNA in the Absence of TATA

Gilfillan¹,

Stelzer²,

Piaia³

et al. 2005

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2⅐TBP with DNA. We show that NC2⅐TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP⅐TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATAcontaining and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2⅐TBP displays a K D for DNA of ϳ2 ؋ 10 ؊9 M for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP⅐DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence.TATA-binding protein (TBP) 1 binds to eukaryotic promoters to nucleate initiation of transcription (1-3). The crystal structure of the TBP⅐DNA complex revealed a saddle-shaped conformation of TBP in which the highly conserved carboxyl terminus forms a concave surface that interacts with the minor groove of DNA. As a consequence, the minor groove becomes dramatically widened, and the DNA is severely bent (4). The characteristic bend as well as the TBP conformation is conserved in the co-crystal structures of TBP⅐TFIIA⅐DNA (5, 6), TBP⅐TFIIB⅐DNA (7), and TBP⅐NC2⅐DNA (8).The thermodynamics and kinetics of TBP-TATA interactions have been investigated in detail. Both yeast and human TBP show specificity for TATA boxes. Values for the K D are in the range of 5 ϫ 10 Ϫ10 to 2 ϫ 10 Ϫ8 M for yeast TBP (9 -13) and 4.6 ϫ 10 Ϫ10 to 1 ϫ 10 Ϫ9 M for human TBP (14, 15). Variations may be accounted for in part by the specific conditions and the different methods employed, which are gel shifts and footprints versus solution FRET studies. Specificities of TBP for TATA range from Ͻ1 order of magnitude if a canonical TATA is compared with single point mutants in the TATA sequence to roughly 3 orders of magnitude relative to random DNA (14). Human TBP has been proposed to bind DNA through the conserved domain in the usual specific manner or, alternatively, in a nonspecific manner that depends on its non-conserved amino-terminal region (16).Several factors modulate binding of TBP to DNA. The TBPassociated factors (TAFs) support binding of TBP specifically to TATA-less promoters via DNA contacts through cognate promoter sequences (17, 18). The general transcription factor TFIIA accelerates and stabilizes binding of TBP to TATA boxes (19 -21). It also functions as a co-activator for some transcriptional activators (22-26). TFIIA further functions as an antagonist of NC2 (also called Dr1/DRAP) (27, 28). Evidence for this equilibrium between NC2 and TFIIA stems from both biochemical investigations in human cells and genetic studies in yeast in which mutants in TFIIA genes w...

“…Inasmuch as TBP has a relatively high affinity for nonspecific DNA (46) and in this capacity can assemble pol II transcription complexes, the cell is likely to employ a number of mechanisms to prevent this nonproductive and potentially detrimental assembly. Our previous studies (26,30,37,39,40) suggest that TBP dimerization represents a physiologically important auto-inhibitory mechanism by which unregulated binding of TBP to DNA is prevented. If so, then there are likely to be mechanisms that dissociate TBP dimers as a prelude to productive binding.…”

Section: Discussionmentioning

confidence: 99%

“…Note that the TBP and SNR6 derivatives are present in a wild type TBP and SNR6 background. Null TBP is a ϩ1 frameshift (40). B, quantitation of 12 replicates of data presented in A in which the median ratio of snr6(⌬BB)/ ⌿-WT, normalized to null TBP, is graphed.…”

Section: Fig 6 Effect Of Tbp Mutants On Pol III Transcriptionmentioning

confidence: 99%

Inhibition of TATA Binding Protein Dimerization by RNA Polymerase III Transcription Initiation Factor Brf1

Alexander¹,

Kaczorowski²,

Jackson-Fisher³

et al. 2004

The Brf1 subunit of TFIIIB plays an important role in recruiting the TATA-binding protein (TBP) to the upstream region of genes transcribed by RNA polymerase III. When TBP is not bound to promoters, it sequesters its DNA binding domain through dimerization. Promoter assembly factors therefore might be required to dissociate TBP into productively binding monomers. Here we show that Saccharomyces cerevisiae Brf1 induces TBP dimers to dissociate. The high affinity TBP binding domain of Brf1 is not sufficient to promote TBP dimer dissociation but in addition requires the TFIIB homology domain of Brf1. A model is proposed to explain how two distinct functional domains of Brf1 work in concert to dissociate TBP into monomers.Eukaryotic genes are transcribed by one of three RNA polymerases, pol 1 I, II, or III. In higher eukaryotes, there are two main pathways of assembling a pol III transcription complex, which are governed in large part by distinct promoter elements located either upstream or downstream of the transcriptional start site (1-3). In the budding yeast Saccharomyces cerevisiae, one major assembly pathway exists, and it involves the binding of TFIIIC to promoter elements internal to tRNA genes. TFIIIC then recruits TFIIIB to a location immediately upstream of the transcriptional start site (4, 5). TFIIIB then directs pol III to the transcriptional start site. TFIIIB is composed of three subunits: the TATA binding protein (TBP), Brf1, and Bdp1 (6 -10).TBP is important for the expression of essentially all eukaryotic genes. It is a saddle shaped protein having aminoand carboxyl-terminal stirrups and a concave/convex surface (11,12). TBP binds to a TATA box promoter element when it is present. However, most pol III genes with an internal promoter lack a TATA box. TBP is delivered to the upstream region of these promoters by Brf1, and Brf1 is recruited by TFIIIC (13-15).The amino-terminal half of Brf1 is homologous to the pol II general transcription factor TFIIB (16 -18). TFIIB binds to TBP along its carboxyl-terminal stirrup, pinning it to DNA (19). In contrast to TFIIB, the TFIIB homology domain of Brf1 has not been shown to independently bind TBP. Where or whether it interacts with TBP is largely unknown. Some evidence places it near the carboxyl-terminal stirrup of TBP (20, 21). The amino-terminal domain of Brf1 interacts with the 131 subunit of TFIIIC as well as the C34 subunit of pol III (14,22). This domain also plays an important role in open complex formation (23,24), the stage at which the template and transcribed strands are separated in preparation for transcription initiation. The carboxyl-terminal half of Brf1 is unrelated to TFIIB but is conserved among Brf proteins. A portion of the carboxyl-terminal half (437-506) of Brf1 binds with high affinity to the convex surface of TBP, snaking along the "top" of TBP and down its amino-terminal stirrup (25).TBP self-associates into dimers and possibly higher order structures through its concave DNA binding surface and carboxyl-terminal stirrup (26 -39). ...