A TATA-binding protein mutant defective for TFIID complex formation in vivo

Abstract

Using an intragenic complementation screen, we have identified a temperature-sensitive TATA-binding protein (TBP) mutant (K151L, K156Y) that is defective for interaction with certain yeast TBP-associated factors (TAFs) at the restrictive temperature. The K151L,K156Y mutant appears to be functional for RNA polymerase I (Pol I) and Pol III transcription, and it is capable of supporting Gal4-activated and Gcn4-activated transcription by Pol II. However, transcription from certain TATA-containing and TATA-less Pol II promoters is reduced at the restrictive temperature. Immunoprecipitation analysis of extracts prepared after culturing cells at the restrictive temperature for 1 h indicates that the K151L,K156Y derivative is severely compromised in its ability to interact with TAF130, TAF90, TAF68/61, and TAF25 while remaining functional for interaction with TAF60 and TAF30. Thus, a TBP mutant that is compromised in its ability to form TFIID can support the response to Gcn4 but is defective for transcription from specific promoters in vivo.

FIG. 1

Isolation and characterization of a TBP mutant that complements a TBP mutant with a Pol III defect. (A) Growth conferred by the indicated TBP derivatives and wild-type (wt) TBP at both 30 and 38°C. Cells were grown overnight in liquid medium, and approximately 10⁵ cells were spotted onto the medium lacking the appropriate markers. (B) Growth conferred by TBP derivatives singly substituted at position 151 or 156. (C) S1 nuclease analysis of tRNA^W transcription with 30 μg of RNA from the indicated TBP derivative after shifting cultures to 38°C for 1 h. (D) Mutant and wild-type strains containing PNOY103 (see text) were grown overnight in liquid medium (2% galactose), spotted to galactose medium, and incubated at 30 and 38°C. (E) S1 nuclease analysis of an unstable portion of the rRNA transcript with 70 μg of RNA from the indicated strains after incubation at 30 or 38°C for one hour.

FIG. 2

Molecular modeling of the K151L,K156Y derivative by using the TBP-DNA cocrystal structure (23) (Rasmol 2.6). (A) TBP is shown as a ribbon drawing, with residues K151 and K156 shown in ball-and-stick format. Known binding sites for DNA, TFIIA, and TFIIB and the C and N termini are indicated (13, 35, 51). (B) Top view of TBP (view in panel A rotated 90° forward).

FIG. 3

The K151L,K156Y mutant is functional for activated transcription at the permissive temperature. wt, wild type. (A) Strains were spotted on plates containing the media indicated at the density shown, followed by incubation at 30°C. Yeast extract-peptone-dextrose (YPD) is used as a growth rate indicator on rich media. Growth on 15 mM AT, galactose, and CuSO4 requires functional interactions with the Gcn4, Gal4, and Ace1 transcription factors, respectively. (B) Analysis of Gcn4-dependent activation of HIS3 transcription. Constitutive HIS3 expression initiates equally from both the +1 and +13 start sites, whereas Gcn4-activated transcription is mediated primarily through the +13 initiation site. Strains were grown to log phase in synthetic complete medium in either the presence (+) or absence (−) of 15 mM AT. Total RNA (30 μg) was hybridized with 100-fold excesses of HIS3 and DED1 probes and then subjected to S1 nuclease digestion. The DED1 transcript is not affected by AT and is used as a loading control in this experiment.

FIG. 4

Transcriptional analysis of the K151L,K156Y strain under restrictive conditions indicates defects for a collection of Pol II-transcribed genes. (A) Mutant and wild-type (wt) TBP-containing strains were grown to log phase and shifted to the restrictive temperature for 1 h. Total RNA isolated before and after the temperature shift was hybridized with a 100-fold excess of the indicated probe and treated with S1 nuclease; tRNA^W served as a loading control. The promoter for the +1 transcript from the HIS3 gene is considered TATA-less, while the promoter for the +13 transcript from the HIS3 gene contains a canonical TATA element. The promoters of the other genes tested (MOT1, CMD1, RPS4, ADH1, PGK1, DED1, ENO2, HTA2, and FUR1) have recognizable TATA elements within 250 bp of their translation start sites. The functional relevance of many of these elements is not currently known. (B) Analysis of cyclin genes at the restrictive temperature. Total RNAs from the indicated strains grown at 30°C and for 1 h at 38°C were blotted to nylon membranes and subsequently hybridized with the indicated cyclin probes. ADH1 served as a loading and transfer control.

FIG. 5

TFIID is disrupted in the K151L,K156Y strain. Coimmunoprecipitation of TFIID from both mutant (K151L,K156Y) and wild-type (wt) TBP-containing strains is shown. Strains were grown to log phase and shifted to the restrictive temperature for 1 h. Whole-cell extracts were prepared before and after the temperature shift, and TFIID was immunoprecipitated with anti-TBP antibodies (α-TBP) coupled to protein A-Sepharose beads. The immunoprecipitated (IP) complexes were separated with either a 7.5 or 12% acrylamide gel and electroblotted to nitrocellulose. Blots were first probed with anti-TBP antibodies to serve as an internal control. Blots were then stripped and reprobed for the indicated TAF antibodies. The load represents 1/10 of the input in the IP lanes.

FIG. 6

Maintenance of activated transcription by Gcn4 under the restrictive conditions in the TFIID-disrupted mutant TBP strain. Strains were grown to log phase in synthetic complete medium, and cultures were shifted to the restrictive temperature for 1 h. Cells were then grown for an additional hour in the presence (+) or absence (−) of 15 mM AT, and total RNA was analyzed for HIS3, DED1, and tRNA expression by S1 analysis. wt, wild type.