TFIIIC determines RNA polymerase III specificity at the TATA-containing yeast U6 promoter.

Roberts, Sadia; Colbert, Trenton; Hahn, Steven

doi:10.1101/gad.9.7.832

Cited by 31 publications

(26 citation statements)

References 46 publications

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“…The homologs TFIIB and BRF have been shown to bind TBP-DNA with about the same affinity (37). However, it is clear from previous work (10,19,22) and the results shown here that BRF binds TBP-DNA by a mechanism very different from that used by TFIIB.…”

Section: Discussioncontrasting

confidence: 54%

“…2A), which is at least threefold higher than the affinity seen for full-length BRF ( Fig. 2A) (29,37). These C-BRF derivatives also recruited BЉ to the complex with the same affinity as full-length BRF (not shown).…”

Section: Resultsmentioning

confidence: 89%

“…Gel and running buffers used for Fig. 1, 2, 3, and 5B are as described previously (37) and contain 1.5 mM (final concentration) magnesium acetate. Gel and running buffers used for Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Cleavage was allowed to proceed for 2 min before addition of 2 l of 50% glycerol. Reactions were loaded onto 1.5 mM (final concentration) magnesium acetate native polyacrylamide gels as described previously (37). Protein-DNA complexes were excised from gels, and the DNA was eluted into 300 l of 90 mM Tris-92 mM boric acid-2.5 mM EDTA at pH 8.3.…”

Section: Methodsmentioning

confidence: 99%

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

Colbert

Lee

Schimmack

et al. 1998

Molecular and Cellular Biology

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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...

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Section: Discussioncontrasting

confidence: 54%

Section: Resultsmentioning

confidence: 89%

“…Gel and running buffers used for Fig. 1, 2, 3, and 5B are as described previously (37) and contain 1.5 mM (final concentration) magnesium acetate. Gel and running buffers used for Fig.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Colbert

Lee

Schimmack

et al. 1998

Molecular and Cellular Biology

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“…The yeast U6 upstream promoter has no specificity for recruitment of Brf1 over TFIIB. Rather, it is the binding of TFIIIC to the downstream site that specifies Brf1-TFIIIB recruitment and Pol III transcription (32). It seems likely that, in human cells, as-yet-unidentified factors associated with either TFIIB, Brf1, TBP, or SNAP c prevent assembly of TFIIB and/or favor assembly of Brf2 into the U6 initiation complex.…”

Section: Vol 25 2005 Structure-function Analysis Of Brf2 9415mentioning

confidence: 99%

Structure-Function Analysis of the Human TFIIB-Related Factor II Protein Reveals an Essential Role for the C-Terminal Domain in RNA Polymerase III Transcription

Saxena

Schramm

et al. 2005

Molecular and Cellular Biology

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The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAP c , a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.In eukaryotes, the job of transcribing nuclear genes is divided among three different RNA polymerases: RNA polymerase I (Pol I), RNA Pol II, and RNA Pol III. A common step in transcription initiation by all three eukaryotic RNA polymerases is the recruitment of the polymerase to the proper promoters by a specific factor or complex. This transcription factor serves as a bridge between promoter-bound factors that nucleate preinitiation complex assembly and the RNA polymerase enzyme itself. For Pol II and Pol III transcription, the factors that accomplish this task are TFIIB and TFIIIB, respectively.Although TFIIB is a single polypeptide, the TFIIIB activity is minimally composed of three polypeptides (see references 11 and 33 for reviews). In human cells, two forms of the TFIIIB complex have been characterized thus far: Brf1-TFIIIB, which functions on type 1 and 2 Pol III promoters, and Brf2-TFIIIB, which functions on type 3 Pol III promoters such as the human U6 small nuclear RNA (snRNA) promoter. Brf1-TFIIIB and Brf2-TFIIIB both contain the TATA-box binding protein TBP and the SANT domain protein Bdp1. They differ by the presence of different members of a family of TFIIB-related factors: Brf1 in Brf1-TFIIIB and Brf2 in Brf2-TFIIIB.TFIIB, Brf1, and Brf2 each contain a Zn ribbon domain at their N terminus, followed by a core domain consisting of two imperfect repeats. In this region, human Brf2 is 20% identical with human TFIIB and 18% identical with human Brf1. In addition, Brf1 and Brf2 contain a C-terminal extension. In Brf1, this C-terminal segment contains two regions called homology domains II and III, which are conserved among yeast and human proteins. Moreover, a third region called homology domain I is conserved among several different yeast species (25...

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A suppressor of mutations in the class III transcription system encodes a component of yeast TFIIIB.

et al. 1996

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Class III genes depend on TFIIIB for recruitment of RNA polymerase III. Yeast TFIIIB is comprised of three components: TBP, TFIIIB70 and a 90 kDa polypeptide contained in the fraction B″. We report the isolation of the yeast gene TFC7 which, based on genetic and biochemical evidence, encodes the 90 kDa polypeptide. TFC7 was isolated as a multicopy suppressor of temperature‐sensitive mutations in the two largest subunits of TFIIIC. It is an essential gene, encoding a polypeptide of 68 kDa migrating with an apparent size of approximately 90 kDa. In gel shift assays, recombinant TFC7 protein (rTFC7) alone did not bind detectably to DNA, or to the TFIIIC‐DNA complex even in the presence of TBP or TFIIIB70, but it was required to assemble the TFIIIB‐TFIIIC‐DNA complex. The two‐hybrid assay pointed to an interaction between TFC7 protein and tau 131, the second largest subunit of TFIIIC (that also interacts with TFIIIB70). rTFC7p can replace the B″ component of TFIIIB for synthesis of U6 RNA in a system reconstituted with recombinant TBP and TFIIIB70 polypeptides and highly purified RNA polymerase III. Surprisingly, specific transcription of the SUP4 tRNATyr gene promoted by rTFC7p was much weaker than with B″. An additional factor activity, provided by the recently identified TFIIIE fraction, was required to restore control levels of transcription.

show abstract

TFIIIC determines RNA polymerase III specificity at the TATA-containing yeast U6 promoter.

Cited by 31 publications

References 46 publications

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

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

Structure-Function Analysis of the Human TFIIB-Related Factor II Protein Reveals an Essential Role for the C-Terminal Domain in RNA Polymerase III Transcription

A suppressor of mutations in the class III transcription system encodes a component of yeast TFIIIB.

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