The archaeal RNA polymerase (RNAP) shares structural similarities with eukaryotic RNAP II but requires a reduced subset of general transcription factors for promoter-dependent initiation. To deepen our knowledge of cellular transcription, we have determined the structure of the 13-subunit DNA-directed RNAP from Sulfolobus shibatae at 3.35 Å resolution. The structure contains the full complement of subunits, including RpoG/Rpb8 and the equivalent of the clamp-head and jaw domains of the eukaryotic Rpb1. Furthermore, we have identified subunit Rpo13, an RNAP component in the order Sulfolobales, which contains a helix-turn-helix motif that interacts with the RpoH/Rpb5 and RpoA′/Rpb1 subunits. Its location and topology suggest a role in the formation of the transcription bubble.

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...

The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins. HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively. Unlike other winged helix-turn-helix proteins, HSF's wing does not appear to contact DNA, as based on a previously solved crystal structure. Instead, the structure implies that the wing is involved in protein-protein interactions, possibly within a trimer or between adjacent trimers. To understand the function of the wing in the HSF DNA-binding domain, a Saccharomyces cerevisiae strain was created that expresses a wingless HSF protein. This strain grows normally at 30 degrees C, but shows a decrease in reporter gene expression during constitutive and heat-shocked conditions. Removal of the wing does not affect the stability or trimeric nature of a protein fragment containing the DNA-binding and trimerization domains. Removal of the wing does result in a decrease in DNA-binding affinity. This defect was mainly observed in the ability to form the first trimer-bound complex, as the formation of larger complexes is unaffected by the deletion. Our results suggest that the wing is not involved in the highly co-operative nature of HSF binding, but may be important in stabilizing the first trimer bound to DNA.