High-resolution mapping of nucleoprotein complexes by site-specific protein-DNA photocrosslinking: organization of the human TBP-TFIIA-TFIIB-DNA quaternary complex.

Abstract

We have used a novel site-specific protein-DNA photocrosslinking procedure to define the positions of polypeptide chains relative to promoter DNA in binary, ternary, and quaternary complexes containing human TATAbinding protein, human or yeast transcription factor IIA (TFIIA), human transcription factor IIB (TFIIB), and promoter DNA. The results indicate that TFIIA and TFIIB make more extensive interactions with promoter DNA than previously anticipated. TATA-binding protein, TFHA, and TFIIB surround promoter DNA for two turns of DNA helix and thus may form a "cylindrical clamp" effectively topologically linked to promoter DNA. Our results have implications for the energetics, DNA-sequence-specificity, and pathway of assembly of eukaryotic transcription complexes.Transcription initiation at a eukaryotic protein-encoding gene requires assembly on promoter DNA of a transcription complex consisting of RNA polymerase II and six general transcription factors: TFIIA, TFIIB, TFIID [or TATA-binding protein (TBP)], TFIIE, TFIIF, and TFIIH (1). The smallest transcription complex generally competent for basal and activated transcription contains at least 35 distinct polypeptides and has a molecular mass in excess of 2 MDa (1). Understanding transcription initiation and transcription activation will require elucidation of the structures and the arrangement of these polypeptides relative to each other and to promoter DNA. Recently, structures have been determined for several polypeptides and polypeptide domains within the transcription complex: i.e., TBP core domain (TBPc), alone and in the TBPc-DNA binary complex (2-6); TFIIA in the TBPc-TFIIA-DNA ternary complex (7,8); TFIIB core domain (TFIIBc), alone and in the TBPc-TFIIBc-DNA ternary complex (9, 10); and TFIIB Nterminal domain (11). However, the fully assembled transcription complex is too large for structure determination by NMR spectroscopic and x-ray crystallographic methods. Therefore, information regarding the arrangement of polypeptides relative to each other and to promoter DNA within the fully assembled transcription complex must come from imaging (12) or biochemical methods (13-15). EXPERIMENTAL STRATEGYIn this work, we have used a novel site-specific protein-DNA photocrosslinking procedure to determine the positions of individual polypeptides relative to promoter DNA within the complex. By "position," we refer to the translational position relative to the transcription start site, the rotational orientation relative to the DNA helix axis, and the groove orientation relative to the DNA major and minor grooves. Our procedure has four steps ( Fig. 1): (i) we use chemical (16-18) and enzymatic (19) reactions to prepare a promoter DNA fragment containing a photoactivatible crosslinking agent and an adjacent radiolabel incorporated at a single defined DNA phosphate; (ii) we form a protein-DNA complex using the site-specifically derivatized promoter DNA fragment, and we UV-irradiate the complex, initiating covalent crosslinking with polypeptide chains in dir...

Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

“…TBP and IIB exhibit patterns of crosslinking in the TBP-IIB-IIF-RNAPII-promoter complex similar to those previously reported for the TBP-DNA and TBP-IIB-DNA complexes (21) and in agreement with the crystallographic structures of the TBP-DNA and TBP-IIB-DNA complexes (16)(17)(18) (Fig. 1D).…”

“…To define positions of polypeptides relative to promoter DNA within the human TBP-IIB-IIF-RNAPII-promoter complex, we have performed site-specific protein-DNA photocrosslinking (ref. 21; Fig. 1).…”

“…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.…”

“…Radiophosphorylated derivatized oligodeoxyribonucleotides (10 pmol), M13 universal sequencing primer (10 pmol; 5Ј-TGACCGGCAGCAAAATG-3Ј), and M13mp18-AdMLP or M13mp19-AdMLP ssDNA (0.4 pmol; ref. 21), were annealed in 40 l of 20 mM Tris⅐HCl, pH 8.0͞25 mM KCl͞4 mM MgCl 2 ͞1 mM EDTA, by using a temperature gradient of 65°C to 25°C in 90 min. They were extended and ligated by the addition of T4 DNA polymerase [3 units; New England Biolabs; desalted into 10 mM potassium phosphate (pH 6.5) and 50% glycerol], T4 DNA ligase (5 units; Boehringer; desalted into 20 mM Tris⅐HCl, pH 7.5͞60 mM KCl͞1 mM EDTA͞5 mM ␤-mercaptoethanol͞50% glycerol), 2Ј-deoxynucleoside 5Ј-triphosphates (40 nmol each; Pharmacia), and ATP (100 nmol; Pharmacia), and incubation for 15 min at 25°C followed by 30 min at 37°C.…”

TFIIA Changes the Conformation of the DNA in TBP/TATA Complexes and Increases their Kinetic Stability

“…3 Studies of TFIIA/TBP/TATA ternary complexes show that TFIIA contacts TBP as well as the DNA upstream and within the TATA box. [31][32][33][34][35][36] With the goal of obtaining a better understanding of the roles that TBP and TFIIA play in transcriptional regulation, we used FRET assays to accurately measure changes in the distance between the ends of the DNA upon binding TBP and TBP/TFIIA. We found that TFIIA enhances the stability of the bent state of TBP/TATA complexes, makes the complexes less sensitive to salt concentration, and causes the conformation of non-consensus TATA DNA bound by TBP to be the same as that of a consensus TATA box.…”