The structural basis for the oriented assembly of a TBP/TFB/promoter complex

Abstract

Recently the definition of the metazoan RNA polymerase II and archaeal core promoters has been expanded to include a region immediately upstream of the TATA box called the B recognition element (BRE), so named because eukaryal transcription factor TFIIB and its archaeal orthologue TFB interact with the element in a sequence-specific manner. Here we present the 2.4-Å crystal structure of archaeal TBP and the C-terminal core of TFB (TFB c) in a complex with an extended TATA-box-containing promoter that provides a detailed picture of the stereospecific interactions between the BRE and a helix-turn-helix motif in the C-terminal cyclin repeat of TFBc. This interaction is important in determining the level of basal transcription and explicitly defines the direction of transcription.T he archaeal transcription preinitiation complex represents a simplified homologue of its eukaryal type II counterpart, requiring only a highly homologous RNA polymerase II (pol II), a typical TATA-box-binding protein (TBP), and transcription factor B (TFB), the homologue of the eukaryal TFIIB (1, 2). Most eukaryal pol II and archaeal transcription preinitiation complexes assemble around an eight-base-pair core element found in nearly all promoters: the TATA box (3-5). The first step in both cases is the binding of TBP to the eight-base-pair TATA box. Crystal structures of both eukaryal and archaeal TBP bound to short TATA-box-containing promoter fragments revealed a highly distorted promoter in which the TATA box was partially unwound and bent about 75-80°toward the major groove, but flanked by normal B-DNA (6, 7). This architectural distortion of the TATA element allows further transcription factors to bind either through stepwise addition or through recruitment of a holoenzyme (8). All crystal structures of eukaryal TBP͞TATA-box complexes reported to date, including ternary complexes with TFIIA or TFIIB, showed TBP bound in the same orientation relative to the start site of transcription, that is with the C-terminal stirrup of TBP upstream of the TATA box (6, 7, 9-12). However, given the nearly perfect symmetry of the TBP͞TATA-box interface, it remained unknown what would specify TBP's orientation relative to the transcription start site and thus determine the polarity of transcription (13). Early experiments indicated that changing the direction of the TATA box alone did not alter the direction of transcription (14, 15). Affinity cleavage experiments have directly confirmed that yeast TBP by itself binds to the TATA box in either orientation, with only a 60:40 preference for the direction seen in the crystal structures (16). Thus, additional factors are required to enforce the unidirectionality of transcription. Parallel studies, one focusing on the eukaryal pol II promoter (17) and the other on an archaeal promoter (18), implicated a consensus sequence element of at least six or seven base pairs contiguous with the upstream end of the TATA box as a binding site for TFB and TFIIB, respectively; the B recognition element, or BRE. Bell e...

Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

“…2 and 3). The results are as expected based on the crystallographic structures of TBP-DNA and TBP-TFBc-DNA complexes (27,28) and on DNase I footprinting experiments with archaeal TBP-DNA and TBP-TFBc-DNA complexes (7,11,13). The results are similar to published results for eukaryal initiation complexes (43,44,59), supporting the homology between the two transcription systems.…”

“…2 and 3). TFB-DNA crosslinking in the TATA-element region is as expected based on crystallographic structures of TBP-TFBc-DNA complexes (27,28) and on footprinting experiments with TBP-TFBc-DNA complexes (7,11,13,60,61). TFB-DNA crosslinking immediately upstream of the TATA element is as expected for sequence-specific interaction between the BH4Ј-BH5Ј helix-turn-helix motif of the TFB Cterminal domain and the TFB recognition element (Refs.…”

“…2 and 3). Based on crystallographic structures of complexes of TBP, TFB C-terminal domain, and DNA (27,28), this crosslinking cannot be ascribed to interactions between the TFB C-terminal domain and DNA.…”

“…Archaeal TFB belongs to the TFIIB family, whose members bind promoter DNA, bind RNAP, and serve as bridges between the TBP-TATA-element complex and RNAP (26 -29). Archaeal TFB has the characteristic domain organization of other TFIIB family members (31, 32), comprising a C-terminal domain (TFBc) that mediates interactions with the TBP-TATA-element complex (7,27,28) and an N-terminal domain (TFBn) that mediates interactions with RNAP (33,34). The TFB N-terminal domain consists of a conserved metal binding region ("zinc ribbon"; Refs.…”

An expanding family of archaeal transcriptional activators

“…Whether the Lrp activation surface that is defined here by gain-of-function mutations is a site of direct interaction with TBP (or TFB) or whether it is involved in Lrp dimer interactions that determine the effective presentation of UAS-bound Lrp to the core transcription apparatus also remains to be determined. The crystal structure of an archaeal TBP-TFB-DNA ternary complex (from Pyrococcus woesei) has been determined (22,23), but the structure of an activator-TBP-TFB-DNA complex remains an important (and possibly arduous) objective. In fact, the determination of crystal structures of promoter-bound transcriptional activators interacting with their target elements in the transcription apparatus is limited to two examples from bacterial transcription: a CAP͞RNAP ␣ subunit C-terminal domain͞DNA complex (24) and a cI protein͞ domain 4͞DNA complex (25).…”