Reconfiguring the connectivity of a multiprotein complex: fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB

Abstract

Transcription factor (TF) IIIB, the central transcription initiation factor of RNA polymerase III (pol III), is composed of three subunits, Bdp1, Brf1 and TATA-binding protein (TBP), all essential for normal function in vivo and in vitro. Brf1 is a modular protein: Its N-proximal half is related to TFIIB and binds similarly to the C-terminal stirrup of TBP; its C-proximal one-third provides most of the affinity for TBP by binding along the entire length of the convex surface and N-terminal lateral face of TBP. A structure-informed triple fusion protein, with TBP core placed between the N- and C-proximal domains of Brf1, has been constructed. The Brf1-TBP triple fusion protein effectively replaces both Brf1 and TBP in TFIIIC-dependent and -independent transcription in vitro, and forms extremely stable TFIIIB-DNA complexes that are indistinguishable from wild-type TFIIIB-DNA complexes by chemical nuclease footprinting. Unlike Brf1 and TBP, the triple fusion protein is able to recruit pol III for TATA box-directed transcription of linear and supercoiled DNA in the absence of Bdp1. The Brf1-TBP triple fusion protein also effectively replaces Brf1 function in vivo as the intact protein, creating a TBP paralogue in yeast that is privatized for pol III transcription.

Fig. 1.

The Brf1n-TBPcore-Brf1c triple fusion can replace Brf1 and TBP for TFIIIC-independent transcription and for formation of heparin-resistant TFIIIB–DNA complexes. (A) A model of the Brf1 (1–382)-TBP core-Brf1 (439–596) triple fusion. DNA (yellow and green sticks), TBPc (blue ribbon), and the resolved Brf1 (439–506) segment (red ribbon) are from ref. . A possible path of a (GS)₆ linker (orange) between TBP residue 240 and Brf1 residue 439 (space-filled) is shown. The Brf1 (76–273) segment modeled into the TFIIB–TBP–DNA crystal structure (8) is also shown (light green) with Brf1 residue 273 space-filled and the TBPc N-terminal residue 61 (space-filled in cyan) highlighted. A cartoon identifying the segments comprising the triple fusion is sketched out below the model [but the (GS)₆ linker is inapparent on this scale]. (B) TFIIIC-independent transcription. Protein–DNA complexes were formed with 50 fmol of supercoiled plasmid DNA (lanes 1–7) or a 364-bp linear DNA fragment (lanes 8–14) and 200 fmol of the TFIIIB components designated above each lane. U6_LboxB transcripts and a labeled DNA recovery marker (rm) are identified on the left. The weak transcripts with lower mobility than r-U6 and l-U6 RNA in lanes 9 and 11 do not depend on either Brf1 or Bdp1 (data not shown, but cf. 34). (C) EMSA. Protein–DNA complexes were formed with 200 fmol of the TFIIIB components designated above each lane and 8 fmol of a 57-bp TATA box-containing probe (specified in Materials and Methods) and were analyzed on a 4% native polyacrylamide gel.

Fig. 2.

The Brf1n-TBPc-Brf1c triple fusion protein is competent for TFIIIC-dependent transcription and formation of a heparin-resistant TFIIIB–DNA complex. (A) TFIIIC-dependent transcription of the SUP4 tRNA gene variant TA-30 (50 fmol) as supercoiled plasmid (lanes 5–8, 11, 12) or 160-bp DNA fragment (lanes 1–4, 9, 10) with 50 fmol of TFIIIC, 100 fmol of Bdp1, and 25 fmol of wild-type Brf1 + TBPc or Brf1n-TBPc-Brf1c, as indicated above each lane. Multiple-round (lanes 1–8) and single-round (lanes 9–12) transcription was performed as described in Materials and Methods. The SUP4 transcript and recovery marker (rm) are identified on the left. (B) EMSA. Protein–DNA complexes were formed with 100 fmol each of TFIIIB component subunits and 50 fmol of TFIIIC on a TA-30 SUP4 DNA probe (1 fmol) as indicated above each lane. TFIIIC–TFIIIB–DNA (CB), TFIIIC–Brf1–TBPc–DNA (CB′), TFIIIC–DNA (C), and heparin-resistant TFIIIB–DNA (B-hep) complexes are identified on the left. (C) Nhp6a suppresses nonspecific transcription. Nhp6a (quantities, in nanograms, indicated above each lane) was mixed with TFIIIB assembled with wild-type Brf1 and TBPc (lanes 1–4) or Brf1n-TBPc-Brf1c (lanes 5–8), followed by the addition of TFIIIC and nonspecific competitor DNA for single-round transcription as specified for A.(D) Quantification of C, averaged with an additional experiment after normalization to the yield of SUP4 RNA with wild-type TFIIIB in the absence of Nhp6a. Average deviations exceeding symbol sizes are shown; the 800-ng Nhp6a points are from a single experiment. Open symbols, nonspecific transcription; filled symbols, specifically initiating transcription; squares, reference type TFIIIB; circles, TFIIIB assembled with Brf1n-TBPc-Brf1c.

Fig. 3.

The Brf1n-TBPc-Brf1c triple fusion and the Brf1n-TBPc/Brf1c split fusion can replace Brf1 function in vivo. Ten-fold serial dilutions of the wild-type (chromosomal BRF1; Chr-BRF) strain DY9876 and strains dependent on plasmid-borne GAL1 promoter expression of Brf1 (pGal-BRF), Brf1n-TBPc-Brf1c (pGAL-TF), and the Brf1n-TBPc/Brf1c split (pGal-Split) were plated on yeast extract/peptone/dextrose or yeast extract/peptone plus galactose (serial dilutions of 10⁷ cells per ml) and grown at 15°C, 30°C, and 37°C, as indicated.

Fig. 4.

Repression of Ile tRNA I(TAT) transcription in response to DNA damage by methylmethane sulfonate (MMS). The steady-state levels of pre-tRNA I(TAT) were measured as a function of time of treatment with MMS by Northern blot analysis. Samples were collected before the addition of MMS and after 0.5, 1, 2, and 4 h of treatment in yeast strains producing Brf1 from the chromosomal (BRF1) gene (Chr-BRF, filled squares) or Brf1 (pGal-BRF1, open triangles), Brf1n-TBPc-Brf1c (pGal-TF, open circles), and the Brf1n-TBPc + Brf1c split (pGal-Split, filled diamonds) from the corresponding genes on centromeric plasmids under control of the GAL1 promoter. The hybridization signal for the pre-tRNA was normalized to the stable pol II transcript U4 and is plotted as fraction of the initial level of pre-tRNA (mean and average deviation of two independent experiments).