Architecture of Protein and DNA Contacts within the TFIIIB-DNA Complex

Abstract

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIBrelated factor (BRF), and B؆. Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B؆ surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Yeast RNA polymerase III (Pol III) is recruited to promoters by the transcription factor TFIIIB (13,14,46). TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF; also termed TFIIIB70), and a third subunit termed BЉ. Together, these three factors form a highly stable protein-DNA complex upstream of the transcription start site at Pol III promoters. This TFIIIB-DNA complex can promote multiple rounds of initiation by polymerase (20). In vivo, TFIIIB is recruited to promoters by the factor TFIIIC (14). In purified systems in vitro, TFIIIB can be positioned at a promoter independent of TFIIIC, provided the promoter contains a functional TATA element (17, 31); an example of such a promoter is the yeast U6 promoter.The preinitiation complexes for RNA Pol II and III and Archaea polymerase are related in several respects. All three complexes contain TBP as an essential component (41,44). In addition, both complexes contain subunits related to the TFIIB family of proteins. The Pol II factor TFIIB contains a Zn binding site at its N terminus and a C-terminal core domain (TFIIBc) containing the TBP and DNA binding activities (1, 32). The Zn binding site of TFIIB is essential for recruitment of Pol II enzyme (3,8,16). The Archaea factor TFB is 32% identical and 56% similar to human TFIIB (34, 36). TFB contains a Zn binding site at its N terminus (48), and the Cterminal core domain of TFB binds TBP-DNA similarly to TFIIB (25). The Pol III factor BRF is homologous to TFIIB and TFB over the Zn binding site and core domain and is about 25% identical and 35% similar to TFIIB and TFB (9,11,30). However, BRF also contains a 30-kDa domain at its C terminus (the C-BRF) which is conserved only among other BRFs and appears to play a major role in interaction with TBP. In two-hybrid and affinity chromatography assays, the C-terminal domain but not the N-terminal domain interacts strongly with TBP (10, 22). In addition, it has been recently shown that the C-BRF alone can form a complex with TBP, BЉ, and DNA (19). The role of the BRF N-terminal domain is not yet clear, although it does...

Self-association of the Amino-terminal Domain of the Yeast TATA-binding Protein

“…The expression vectors used for protein purification and pull-down assays were pPROTet.E (2.2 kb, Cam R , from Clontech) encoding an amino-terminal (His-Asn) 6 affinity tag for in-frame fusion with target genes and pFLAG.mac (5 kb, Amp R from Sigma) expressing an aminoterminal FLAG epitope tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) for inframe fusion with target genes. These were maintained on LB agar containing 34 g/ml chloramphenicol and 50 g/ml ampicillin, respectively.…”

“…Samples were prepared by mixing 20 g of purified (His-Asn) 6 tagged amino-terminal protein with crude cell lysate proteins (1 mg) containing FLAG-tagged amino-terminal domain in a reaction volume of 800 l. The incubation buffer was 94 mM sodium phosphate (pH 7.0 at 20°C), 6.6 mM sodium acetate, and 222 mM NaCl. Samples were incubated at 4°C for 3 h. Cobalt affinity resin was equilibrated with 50 mM sodium phosphate (pH 7.0 at 20°C), 300 mM NaCl.…”

“…After incubation for 8 h at 30°C, cells were collected by centrifugation and lysed at 4°C with lysozyme (0.75 mg/ml) followed by sonication (3 ϫ 30 s with a pause for 30 s on ice between each burst). The (His-Asn) 6 -tagged TBP amino-terminal domain was purified by absorption on cobalt affinity resin (Talon, Clontech) followed by elution with 50 mM sodium acetate, 300 mM NaCl, pH 5.0 as specified by the manufacturer. Samples were concentrated using Centricon YM3000 filter devices (Millipore), and pH was adjusted to 7.0 with sodium phosphate buffer.…”

“…Protein Cross-linking-Extracts were prepared from E. coli BL21 Star BEM cells expressing (His-Asn) 6 -tagged TBP amino-terminal domain pROTet.E.N1 or from cells containing the empty control vector pPROTet.E by the lysozyme-sonication method. (His-Asn) 6 -tagged proteins were partially purified by cobalt affinity chromatography as described above.…”

“…Together with TBP-associated protein factors (TAFs (3,4)), it forms multisubunit complexes required for transcription by RNA polymerases I, II, and III (2,(5)(6)(7). TBP is a two-domain protein with a strongly conserved carboxyl-terminal domain (ϳ80% sequence identity among eukaryotes (8,9)) and an amino-terminal domain highly divergent in both length (18 -173 residues) and sequence (8, 10 -12).…”