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An Activation-Specific Role for Transcription Factor TFIIB in Vivo (Sua7͞pho4͞pho5͞transcriptional Control)

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An Activation-Specific Role for Transcription Factor TFIIB in Vivo (Sua7͞pho4͞pho5͞transcriptional Control) Proc. Natl. Acad. Sci. USA Vol. 96, pp. 2764–2769, March 1999 Biochemistry An activation-specific role for transcription factor TFIIB in vivo (SUA7yPho4yPHO5ytranscriptional control) WEI-HUA WU AND MICHAEL HAMPSEY* Department of Biochemistry, Division of Nucleic Acids Enzymology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635 Edited by Robert G. Roeder, The Rockefeller University, New York, NY, and approved January 20, 1999 (received for review November 9, 1998) ABSTRACT A yeast mutant was isolated encoding a sin- a study of PIC formation and transcriptional activity demon- gle amino acid substitution [serine-53 3 proline (S53P)] in strated that PIC assembly occurs by at least two steps and that transcription factor TFIIB that impairs activation of the the TATA box and TFIIB also can affect transcription subse- PHO5 gene in response to phosphate starvation. This effect is quent to PIC assembly (11). Thus, processes other than factor activation-specific because S53P did not affect the uninduced recruitment are potential activator targets. level of PHO5 expression, yet is not specific to PHO5 because TFIIB is recruited to the PIC by the acidic activator VP16 Adr1-mediated activation of the ADH2 gene also was impaired and the proline-rich activator CTF1 (3, 12). VP16 directly by S53P. Pho4, the principal activator of PHO5, directly targets TFIIB (3) and this interaction is required for activation interacted with TFIIB in vitro, and this interaction was (13, 14). Interestingly, VP16 induces a conformational change impaired by the S53P replacement. Furthermore, Pho4 in- in TFIIB that disrupts the intramolecular interaction between duced a conformational change in TFIIB, detected by en- the N- and C-terminal domains in solution (15). However, it is hanced sensitivity to V8 protease. The S53P replacement also not known whether the VP16 effect on TFIIB conformation impaired activation of a lexA(op)-lacZ reporter by a LexA fusion affects transcription or if this effect is physiologically signifi- protein to the activation domain of Adr1, thereby indicating cant. Another study questioned the functional significance of that the transcriptional effect on ADH2 expression is specific the VP16-TFIIB interaction (16). Furthermore, the idea that to the activation function of Adr1. These results define an TFIIB recruitment is, in general, a rate-limiting step for activation-specific role for TFIIB in vivo and suggest that transcriptional activation has been challenged by TBP and certain activators induce a conformational change in TFIIB as TFIIB mutations that are defective for TBP-TFIIB-DNA part of their mechanism of transcriptional stimulation. complex formation in vitro, yet respond normally to activators in vivo (17, 18). Transcriptional activators stimulate gene expression by bind- In the yeast Saccharomyces cerevisiae, TFIIB is encoded by ing enhancer elements and contacting, either directly or the essential SUA7 gene (19). Yeast TFIIB is a monomeric indirectly, components of the RNA polymerase II (RNAPII) protein of 345 aa that is structurally similar to human TFIIB, transcriptional machinery. There is considerable evidence that including a zinc binding motif near the N terminus and two activation can occur by a recruitment mechanism (reviewed in imperfect repeats encompassing the C-terminal two-thirds of ref. 1). Accordingly, activators bind general transcription the molecule. The C-terminal domain forms a protease- factors (GTFs) to facilitate formation of the preinitiation resistant core (cIIB) (20, 21) that interacts with the N-terminal complex (PIC). This idea is supported by direct contact in vitro domain (nIIB) (15). In addition to the zinc binding domain, between activators and GTFs, including TATA box-binding nIIB includes a phylogenetically conserved sequence that links protein (TBP) (2), TFIIB (3), TFIIF (4), TFIIH (5), as well as the zinc binding domain to cIIB (22). A solution structure for the Rpb5 subunit of RNAPII (6), and TBP-associated factors human cIIB (23), and a cocrystal structure for a TATA-TBP- (7, 8). Furthermore, artificial connections between DNA cIIB ternary complex (24), revealed that the two repeat binding domains and either GTFs or components of the domains are arranged in pseudo-dyad symmetry, each com- RNAPII holoenzyme complex stimulate transcription (re- posed of five a-helices. The structure of free cIIB is compa- viewed in ref. 1), yet tethering of an activation domain to GTFs rable to the cocrystal structure, except that free cIIB is more or RNAPII components fails to stimulate transcription (9). compact and the relative orientation of the two repeats is Thus, artificial recruitment of the RNAPII machinery to different, suggesting that TFIIB undergoes a conformational promoter DNA bypasses the activator requirement. change on assembly into the TATA-TBP-TFIIB complex (25). Although a compelling argument can be made for activation A tertiary structure for full-length TFIIB is not available, by recruitment, it does not account for activation of all genes. although an NMR structure for the N-terminal region of Other potential activation mechanisms include activator- Pyrococcus TFIIB revealed that the metal binding motif forms induced conformational changes to either the promoter or to a zinc ribbon similar to that of the elongation factor TFIIS (26). components of the PIC, stabilization of the PIC, covalent In an effort to elucidate the role of TFIIB in transcriptional modification of the PIC, and counteractive effects on the activation, we have generated and characterized a collection of repressive function of chromatin. An especially informative yeast sua7 mutants that encode altered forms of TFIIB. Here case is the Escherichia coli glnA gene, which is activated by we describe one of those mutants, which encodes a serine-53 ATP-dependent, NtrC-mediated isomerization of a s54- to proline (S53P) replacement adjacent to the TFIIB zinc holoenzyme complex from a closed to an open promoter ribbon domain. This replacement impairs activation of specific complex (reviewed in ref. 1). In another case, the Epstein–Barr virus ZEBRA protein stimulates transcription by isomeriza- This paper was submitted directly (Track II) to the Proceedings office. tion of the TATA-TBP-TFIIA ternary complex (10). Recently, Abbreviations: TF, transcription factor; S53P, serine-53 3 proline; RNAPII, RNA polymerase II; PIC, preinitiation complex; TBP, The publication costs of this article were defrayed in part by page charge TATA box-binding protein; cIIB, C-terminal core domain of TFIIB; nIIB, N-terminal domain of TFIIB; YPD, yeast extractypeptoney payment. This article must therefore be hereby marked ‘‘advertisement’’ in dextrose; GST, glutathione S-transferase. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. e-mail: hampsemi@ PNAS is available online at www.pnas.org. umdnj.edu. 2764 Downloaded by guest on September 28, 2021 Biochemistry: Wu and Hampsey Proc. Natl. Acad. Sci. USA 96 (1999) 2765 genes in vivo and adversely affects TFIIB interaction with a expressed in E. coli strain BL-21. After 2.5-hr induction in the cognate activator in vitro. Moreover, the transcriptional defect presence of 0.5 mM isopropyl b-D-thiogalactoside, cells ex- is specific to the activation function of the trans-activator. tracts were prepared and incubated with glutathione Sepha- These results define an activation-specific role for TFIIB in rose-4B beads (Amersham Pharmacia) in buffer A (20 mM vivo. TriszHCl, pH 7.6y0.2 mM EDTAy1mMDTTy1 mM PMSFy1 M NaCly0.1% NP-40), followed by three washes with buffer A z y MATERIALS AND METHODS and three washes with buffer C (20 mM Tris HCl, pH 7.6 0.2 mM EDTAy1mMDTTy1 mM PMSFy100 mM NaCly20% Yeast Strains. The two isogenic strains used in this study glycerol). Fusion proteins were eluted in three times 500-ml were generated as follows. The SUA7 gene, carried on a CEN elution buffer (10 mM reduced glutathione in 50 mM TriszHCl, HIS3 vector, was mutagenized by PCR under error-prone pH 8.0). His6-tagged proteins were purified as described by conditions (27) and introduced into yeast strain YIP91–13B Qiagen. [MATa CYC1 his3-D1 leu2–3, 112 trp1–289 ura3–52 ade1–100 m 1 For GST pull-down experiments, 3 g of fusion protein was sua7::LEU2 (CEN-SUA7-URA3)] (28). His transformants incubated with 20-ml bed volume of glutathione-Sepharose were cured of the CEN-SUA7-URA3 plasmid, selecting for beads in 500 ml of buffer C containing 0.05% NP-40. Input r r strains resistant to 5-fluoroorotic acid (FOA ). FOA strains protein (103) was added and incubated at 4°C for at least 4 hr. were scored for conditional growth phenotypes (28) and Beads were collected by centrifugation, washed three times subsequently scored for phenotypes that often are associated with buffer C containing 500 mM NaCl, and boiled in 20 mlof with transcriptional defects (29). Strain YMH300 harbors loading buffer. Proteins were resolved by SDSyPAGE, trans- plasmid pM507 (sua7–36 HIS3 CEN), which encodes the S53P ferred to nitrocellulose, and probed by Western blot using derivative of TFIIB. Strain YMH130 carries plasmid pM299 rabbit polyclonal antiserum directed against yeast TFIIB (28). (SUA71 HIS3 CEN) and is otherwise identical to YMH300. D Antigen-antibody complex was detected by chemilumines- Strains YMH506 [MATa CYC1 his3 1 leu2–3, 112 trp1–289 cence using goat anti-rabbit IgG conjugated to horseradish ura3–52 ade1–100 sua7::LEU2 (SUA7 TRP1 CEN)] and peroxidase. sua7–36 TRP1 CEN) YMH507 ( are isogenic strain pairs Limited Protease Digestion of TFIIB. Limited V8 protease comparable to YMH130 and YMH300, but include different digestion of wild-type and S53P TFIIB was done as described markers. (15) with minor modifications. In each reaction, 50 ng of Growth Media. YPD medium consists of 1% yeast extract, His6-TFIIB or His6-TFIIB(S53P) was incubated with 0, 5, or 2% peptone, and 2% dextrose.
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