TBP-bound DNA

Local Conformational Properties and Analysis. Overall, TBP-bound DNA is found to exhibit low twist and positive roll, the first and last basepair steps have high rise, as a consequence of the insertion of Phe residues from TBP. To look for the appearance of such specific local properties in the simulations, all the basepair step conformations generated in the simulations were scanned to identify those steps that have average values biased towards these characteristics. RY steps (R stands for purine and Y for pyrimidine) were thus found to share the properties of low twist and positive roll, while YR steps were found to have high rise. 

Figure 28-13 (A) Stereoscopic ribbon drawing of the phyloge-netically conserved 180-residue C-terminal portion of the TATA-binding protein (TBP) from Arabidopsis thaliana. The sequence consists of two direct repeats, giving the protein an approximate twofold symmetry. From Nikolov et al.337 (B) Structure of the corresponding C-terminal core (residues 155-335) of the human TATA-binding protein (TBP) bound to the TATA sequence of a promoter in adenovirus DNA. From Nikolov et al.327 (C) Structure of human transcription factor IIB bound to a TBP from Arabidopsis thaliana, which, in turn, is bound to an adenovirus TATA sequence. Hypothetical B DNA extensions have been modeled at both ends of the DNA segment. The +1 at the left end is the transcription start site and the —43 upstream end is to the right. From Nikolov et al.338 Courtesy of Stephen K. Burley.

The entire top side of TBP bound to the TATA-box DNA is out of the way and is fi e to interact with other factors. This leaves a generous surface on TBP available for interactions with the multitude of factors which are parts of the transcription initiation complex. The same is true for the TFIIA/TFIIB-TBP-complexes. The extensive surfaces displayed by the TFIIA/TFIIB-TBP-DNA complexes represent potential sites for binding basal initiation factors, signal-responsive transcriptional activators, co-activators and mediators, and, most importantly, leave room for Pol II. 

TBP bound to the TATA box is the heart of the initiation complex (see Figure 28.19). The surface of the TBP saddle provides docking sites for the binding of other components (Figure 28.21). Additional transcription factors assemble on this nucleus in a defined sequence. TFIIA is recruited, followed by TFIIB and then TFIIF—an ATP-dependent helicase that initially separates the DNA duplex for the polymerase. Finally, RNA polymerase II and then TFIIE join the other factors to form a complex called the basal transcription apparatus. Sometime in the formation of this complex, the carboxyl-terminal domain of the polymerase is phosphorylated on the serine and threonine residues, a process required for successful initiation. The importance of the carboxyl-terminal domain is highlighted by the finding that yeast containing mutant polymerase II with fewer than 10 repeats is not viable. Most of the factors are released before the polymerase leaves the promoter and can then participate in another round of initiation. 

A key contribution of molecular dynamics simulations to the imderstanding of mechanisms of selectivity and affinity in TBP-DNA complexes is the discovery of the active role of TBP in the formation of the complex. The view derived from crystal structures was that of a passive role for the TBP which only imposed a steric constraint on DNA shape. It appears now from the simulations that TBP can respond to the dynamics of the bound DNA sequence by adjusting its interdomain geometry, and this might be relevant for the construction of the final preinitiation complex. Furthermore, many of the contacts characterized in the crystal structures were found in the simulations to have an important dynamic component, as side chains switch rotamers rather frequently. This conformational freedom makes it possible for TBP to achieve suitable binding contacts with a variety of DNA moieties in a dynamic mode which contributes to enthalpic stabilization. However, the extent of preservation of side chain dynamics in the complex is dependent on the local structure. As it reduces the entropy loss upon complex formation, it provides an additional source of sequence-dependent gain in affinity that is revealed for the first time from the results of the molecular dynamics simulations. 

The proposed structure of TBP bound to DNA based on X-ray crystallography data. (A) A monomer of TBP appears to form a protein "saddle" that sits atop the DNA helix. (B) TBP-binding to the TATA box causes a bend in the DNA double helix. 

The biochemical structure of yeast TBP bound to DNA has been determined by X-ray crystallography and two very interesting observations have been made. The first is that TBP has an internal pseudo-dimer interaction between two adjacent protein domains, forming a "saddle" structure that binds DNA (Figure 24. 7). The exposed surface of TBP in this configuration serves as a contact area for a variety of other transcription factors. 

Since these proteins bind to a specific DNA sequence it is quite natural to compare cognate complexes with complexes in which either the protein or the DNA has been altered, and such comparisons are also undertaken in simulations of hormone receptors (variations of protein and DNA), restriction endonucleases (variation of the DNA), and the TBP in which TBPs bound in different orientation to the same DNA sequence are compared. In this group of studies the nuclear hormone receptors have attracted most of the attention, with several simulations presented for both the glucocorticoid and the estrogen receptors. 

The general transcription factor TFllD is believed to be the key link between specific transcription factors and the general preinitiation complex. However, the purification and molecular characterization of TFllD from higher eucaryotes have been hampered by its instability and heterogeneity. All preparations of TFllD contain the TATA box-binding protein in combination with a variety of different proteins called TBP-associated factors, TAFs. When the preinitiation complex has been assembled, strand separation of the DNA duplex occurs at the transcription start site, and RNA polymerase II is released from the promoter to initiate transcription. However, TFIID can remain bound to the core promoter and support rapid reinitiation of transcription by recruiting another molecule of RNA polymerase. 

DNA binding by TBP is strongly dependent on the presence of T-A base pairs in the TATA box. Bending allows remote sites on the DNA, with their bound cognate specific transcription factors, to come close together such that the proteins can interact to form the transcriptional preinitiation complex. 

TBP binds to the TATA box in the minor groove of DNA (most transcription factors bind in the major groove) and causes an approximately 100-degree bend or kink of the DNA helix. This bending is thought to facilitate the interaction of TBP-associated factors with other components of the transcription initiation complex and possibly with factors bound to upstream elements. Although defined as a component of class II gene promoters, TBP, by virtue of its association with... 

Interaction of the TATA-box-binding protein (TBP) with promoter DNA is rather inefficient and appears to be the rate-limiting step for the start of transcription. TBP must actually dissociate first from the TFIID complex before it can bind to the TATA-box DNA. Dissociation of TBP is facilitated by the dimeric structure of TFIID, when it is not bound to DNA, and by the interaction of TBP with TFIIA. 

The crystal structure of a human TATA-box-binding protein (hTBP), complexed with TATA-box DNA, may be compared with that of a yeast TBP/TATA-box complex and a similar isoform of TBP firom Arabidopsis thaliana, bound to a TATA box in an adenoviral p>romoter (Plate 19).25-27... 

There is a large body of experimental evidence which has been interpreted to fit one or more of these models, but there is still no consensus, in part owing to the different experimental conditions. The most recent data suggest that bending and binding occur at the same time, and that there appear to be two intermediates [60]. Also, correctly pre-bent [61] or more flexible DNA is bound better by TBP, and dissociates more slowly [8,62]. A structural model for these intermediates would serve well in the discrimination of alternatives. 

In section 2.1 the contact interface between TBP and the minor groove of DNA was characterized as anhydrous. This is a common characteristic in all the TBP-DNA complexes available to date. As TBP presents a primarily hydrophobic surface to DNA, most of the hydrogen bond donors and acceptors at this surface are not satisfied by the complexation. Hence, there is likely to be an enthalpic penalty associated with the dehydration of this surface. This penalty is compensated by the favorable increase in entropy associated with the liberation of the surface-bound water molecules into bulk solution. Following this reasoning, there are two aspects of hydration that could contribute to the determination of sequence specificity the ideal sequence would be one which coordinates a large number of water molecules, but binds them least tightly. 

Replication ends when the replication forks meet on the other side of the circular chromosome at the termination site, the ter (t) region. The ter region is composed of a pair of 20-bp inverted repeat ter sequences separated by a 20-bp segment. Each ter sequence prevents further progression of one of the replication forks when a 36-kD ter binding protein (TBP) is bound. How the two daughter DNA molecules separate is not understood, although a type II topoisomerase is believed to be involved. 

Detailed biochemical studies revealed how the Pol II preinitiation complex, comprising a Pol II molecule and general transcription factors bound to a promoter region of DNA, is assembled. In these studies DNase I footprinting and electrophoretic mobility shift assays were used to determine the order in which Pol II and general transcription factors bound to TATA-box promoters. Because the complete, multisubunit TFIID is difficult to purify, researchers used only the isolated TBP component of this general transcription factor in these experiments. Pol II can initiate transcription in vitro in the absence of the other TFIID subunits. 

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