The zinc ribbon domains of the general transcription factors TFIIB and Brf: conserved functional surfaces but different roles in transcription initiation

Abstract

The function of the conserved zinc-binding domains in the related Pol II- and Pol III-specific factors TFIIB and Brf was investigated. Three-dimensional structure modeling and mutagenesis studies indicated that for both factors, the functional surface of the zinc ribbon fold consists of a small conserved patch of residues located on one face of the domain comprised mainly of the second and third antiparallel beta strands. Previous studies have shown that the TFIIB zinc ribbon is essential for recruitment of Pol II into the preinitiation complex. In contrast, Pol III recruitment assays and in vitro transcription demonstrate that the disruption of the Brf zinc ribbon does not lead to a defect in Pol III recruitment but, rather, a defect in open complex formation. Therefore, the same conserved surface of the zinc ribbon domain has been adapted to serve distinct roles in the Pol II and Pol III transcription machinery.

Figure 1

The TFIIB family and the conserved zinc ribbon fold. (A) The three classes of TFIIB family members; (B) schematic of the TFIIB family amino-terminal domain; (C) sequence alignment of Brf, TFB, and TFIIB family members. Conserved secondary structural elements are indicated. The numbering at the top is for Saccharomyces cerevisiae BRF and numbering at the bottom is for S. cerevisiae TFIIB. The models derived in this study were for S. cerevisiae Brf residues 3–33 and TFIIB residues 23–53.

Figure 2

Structure modeling of the Brf and TFIIB zinc ribbons. (A) Polypeptide backbone of two P. furiosis TFB zinc ribbon domains used as templates for modeling (blue) and the backbone of the three best Brf models (pink). This as well as other data described in this paper can be viewed as three-dimensional models at the WWW site indicated in the text. (B) Cartoon of one Brf model with the three β strands indicated. The side chains of four Cys residues (yellow and green) coordinating the zinc atom (red) are shown. (C) Polypeptide backbone of two P. furiosis TFB zinc ribbon domains used as templates for modeling (blue) and the backbone of the three best TFIIB models (pink).

Figure 3

In vivo phenotypes of Brf and TFIIB zinc ribbon mutations and their position on the structure models. (A,B) Two faces of the Brf ribbon structure model are shown with the side chains targeted for mutagenesis color coded by growth phenotype caused by radical substitution. (Yellow) No growth defect; (blue) lethal; (red) temperature sensitive and/or slow growth phenotype. Also indicated are mutations that cause the in vivo phenotypes. (C,D) Two faces of the TFIIB ribbon structure model are shown with the side chains targeted for mutagenesis color coded by in vivo phenotype as was done in A and B. The views of the Brf model in A and B correspond to the same faces of the TFIIB model shown in C and D, respectively. These data can also be viewed three-dimensionally at the web site indicated in the text.

Figure 4

Molecular surface and electrostatic potential of the Brf and TFIIB zinc ribbon functional surfaces. Surface and potential indicated using Grasp (Nicholls et al. 1991). Positive potential, (blue); negative potential, (red); hydrophobic, (white).

Figure 5

In vitro transcription defects caused by Brf zinc ribbon mutations. (A) In vitro transcription was performed for 30 min as described in Materials and Methods using the indicated promoter contained on a supercoiled plasmid. A total of 10 ng of the indicated Brf was used to supplement the Brf W107R extract as indicated. For the U6 (single) panel, preinitiation complexes were performed in the absence of nucleotides for 30 min followed by addition of nucleotides for 3.5 min to limit transcription to a single round. For LEU3 and U6 products, the top arrow indicates the full-length product; the bottom arrow, a processed product derived from full-length transcript. (B) Whole cell extracts were made from the indicated Brf mutant strains grown at 25°C and used for in vitro transcription at 32°C. Complexes were formed in the absence of NTPs for 30 min followed by NTP addition for 3.5 min.

Figure 6

Isolation of preinitiation complexes using immobilized promoter template. (A) Cartoon of the immobilized U6 and mutant templates used. (B) Western blot of complexes formed on wild-type and mutant U6 templates probed with Tfc4 or Rpc34 antisera. Brf was added to the Brf W107R extract as indicated. (C) in vitro transcription activity of isolated PICs. Complexes were formed as in B, except that after washing, nucleotides were added for 5 min and RNA products analyzed by PAGE.

Figure 7

Open complex defect in the Brf zinc ribbon mutants. PICs were formed on 100 ng of supercoiled plasmid DNA as in Fig. 5 (U6 single) using the Brf W107R extract supplemented with 15 ng of the indicated Brf proteins (lanes 1–4). In lane 5, extract made from wild-type yeast was supplemented with 15 ng of wild-type Brf. KMnO₄ was added to 10 mm for 1 min, followed by DNA isolation and detection of modified DNAs on the nontranscribed strand by primer extension. The arrow marks the position of base T −5.