Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

Abstract

By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, Ϸ240 Å channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of Ϸ50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA.Transcription initiation at a eukaryotic protein-encoding gene involves assembly on promoter DNA of a complex consisting of RNA polymerase II (RNAPII) and six general transcription factors: IIA, IIB, IID (or TATA-element binding protein, TBP), IIE, IIF, and IIH (1-3). A subcomplex containing TBP, IIB, IIF, RNAPII, and promoter DNA is stable (4, 5), and, under certain conditions (e.g., conditions that promote DNA melting), is fully competent for transcription initiation (6-11). Results of DNA footprinting experiments indicate that the TBP-IIB-IIF-RNAPII-promoter complex with linear DNA at 30°C involves interactions both upstream and downstream of the transcription start (positions Ϫ42 to ϩ17; H. Lu and D.R., unpublished data) and involves unmelted DNA (11). Thus, the TBP-IIB-IIF-RNAPII-promoter complex with linear DNA at 30°C appears to correspond to the RNA polymerase-promoter ''intermediate complex'' characterized in studies of Escherichia coli RNA polymerase (RP i or RP c2 ; refs. 12-15).The TBP-IIB-IIF-RNAPII-promoter complex contains at least 14 distinct polypeptides (one in TBP, one in IIB, two in IIF, and at least 10 in RNAPII) and has a molecular mass in excess of 700 kDa (1-3). High-resolution structures have been determined for the TBP-DNA and TBP-IIB-DNA complexes (16-18). However, the TBP-IIB-IIF-RNAPII-promoter complex is too large for high-resolution structure determination by current methods. Therefore, information about the structure of the TBP-IIB-IIF-RNAPII-promoter complex must rely on low-resolution structure determination (19,20) supplemented by biochemical and imaging data.In the work in this report, we have used site-specific protein-DNA photocrosslinking and electron microscopy to define protein-DNA interactions within the human TBP-IIB-IIF-RNAPIIpromoter complex. MATERIALS AND METHODSDerivatized Promoter DNA Fragments. Derivatized promoter DNA fragments were prepared essentially as in ref. 21. Oligodeoxyribonucleotides containing phosphorothioate 5Ј to the third nucleotide were synthesized by using solid-phase ␤-cyanoethylphosphoramidite chemistry and tetraethylthiuram disulfide (Applied Biosystems), purified on OPC (Applied Biosystems), and derivatized with azidophenacyl bromide (Sigma; ref. 22). Derivatized oligodeo...

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

“…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.…”

“…1A; Refs. [43][44][45][46][47] as follows: (i) Chemical and enzymatic reactions were used to prepare a DNA fragment containing a phenyl azide photoactivable crosslinking agent ("probe") and an adjacent 32 P radiolabel incorporated at a single, defined DNA phosphate (with a 9.7-Å linker between the reactive atom of the probe and the phosphorus atom of the phosphate and with an ϳ11-Å maximum "reach" between potential crosslinking targets and the phosphorus atom of the phosphate). (ii) The multiprotein-DNA complex of interest was formed using the site-specifically derivatized DNA fragment, and the multiprotein-DNA complex was UV-irradiated, initiating covalent crosslinking with polypeptides in direct physical proximity to the probe.…”

“…Identical results were obtained using an alternative procedure in which complications due to crosslinking within nonspecific and nonproductive complexes were avoided by use of immobilized templates and stringent washing (see the procedures described in Kim et al (45,46)). 3 As a direct control for specificity of crosslinking, three derivatized promoter DNA fragments with probes well outside the range of reported protein-DNA interactions in the eukaryal initiation complex (44,45), i.e. at positions Ϫ70 and ϩ45 on the nontemplate strand and Ϫ66 on the template strand, were constructed and analyzed.…”

“…In published work, systematic site-specific protein-DNA photocrosslinking has been used to characterize the structural organization of eukaryal transcription initiation complexes (43)(44)(45), bacterial transcription initiation complexes (46,47), and the part of an archaeal transcription initiation complex at and downstream of the transcription start site (positions Ϫ1 to ϩ20 of the template strand (21)). Here, we report the use of systematic site-specific protein-DNA photocrosslinking to define the structural organization of the part of an archaeal transcription initiation complex upstream of the transcription start site (positions Ϫ40 to ϩ1; nontemplate and template strands), the part that contains binding determinants for RNAP subunits and general transcription factors.…”

Structure and mechanism of the RNA polymerase II transcription machinery

“…One complication in this proposal is that it does not seem to agree with protein-DNA crosslinking results that show a close overlap between promoter sequences upstream and downstream of the TATA contacted by both TFIIB and TFIIF 96,97,99 . This again suggests the possibility that the arrangement of the general factors on Pol II could change in higher order assemblies with DNA, a possibility that can be addressed by further structural and biochemical studies.…”