The Dr1/DRAP1 heterodimer is a global repressor of transcription in vivo

Abstract

A general repressor extensively studied in vitro is the human Dr1͞DRAP1 heterodimeric complex. To elucidate the function of Dr1 and DRAP1 in vivo, the yeast Saccharomyces cerevisiae Dr1͞DRAP1 repressor complex was identified. The repressor complex is encoded by two essential genes, designated YDR1 and BUR6. The inviability associated with deletion of the yeast genes can be overcome by expressing the human genes. However, the human corepressor DRAP1 functions in yeast only when human Dr1 is coexpressed. The yDr1͞Bur6 complex represses transcription in vitro in a reconstituted RNA polymerase II transcription system. Repression of transcription could be overcome by increasing the concentration of TATA-element binding protein (TBP). Consistent with the in vitro results, overexpression of YDR1 in vivo resulted in decreased mRNA accumulation. Furthermore, YDR1 overexpression impaired cell growth, an effect that could be rescued by overexpression of TBP. In agreement with our previous studies in vitro, we found that overexpression of Dr1 in vivo also affected the accumulation of RNA polymerase III transcripts, but not of RNA polymerase I transcripts. Our results demonstrate that Dr1 functions as a repressor of transcription in vivo and, moreover, directly targets TBP, a global regulator of transcription.Initiation of transcription by RNA polymerase II (RNAPII) is an intricate process requiring different families of transcription factors operating at the promoter (1, 2). One family of factors, the so-called general transcription factors (GTFs), functions to deliver RNAPII to the promoter (for review see ref.3). This process is initiated by association of the TATA-element binding protein (TBP) subunit of TFIID with the TATA motif. TBP recognizes the minor groove of the 8-bp TATA element (4-6), and the TATA element is molded to follow the curved ␤-sheet on the underside of the TBP saddle (6). As a result, the TATA sequence is partially unwound and bent in a smooth arc. The dramatic distortion of the TATA element by TBP allows TFIIB to interact with the phosphodiester backbone of DNA both upstream and downstream of the TATA sequence. The crystal structure of the TBP-TFIIB-DNA ternary complex (TB complex) illustrates how TFIIB recognizes the preformed TBP-DNA complex (7). As suggested by footprinting (8) and crosslinking (9) experiments, TFIIB binds underneath and on one face of the TBP-DNA complex where it interacts with TBP and DNA. TBP-TFIIB contacts are mainly between the basic amino-terminal repeat of TFIIB and the acidic carboxyl-terminal stirrup of TBP, in agreement with mutagenesis studies (10, 11). The TB complex provides the recognition site for entry of RNAPII, which is escorted to the promoter by TFIIF (1-3). The resulting DNA-protein complex (TBPolF) is recognized by TFIIE, providing the recognition site for entry of TFIIH (1-3), resulting in the formation of a transcription competent complex. An alternative model for the formation of transcription complexes has been suggested. In this model the RNAPII ...

Specific Defects in Different Transcription Complexes Compensate for the Requirement of the Negative Cofactor 2 Repressor in Saccharomyces cerevisiae

“…This finding prompted us to determine whether overexpression of rpb2t was also able to suppress the growth defect shown by bur6 single mutants. To analyze the suppression of the bur6 mutation by rpb2t, we used a strain in which the BUR6 promoter was replaced by the GAL10 promoter (P GAL10 -BUR6), rendering cells unable to grow in glucose, since the BUR6 gene is essential for cell growth (Kim et al 1997). As shown in Figure 1A, overexpression of rpb2t allowed the P GAL10 -BUR6 strain to grow in dextrose.…”

“…NC2 consists of two subunits, NC2a (DRAP1) and NC2b (Dr1), which form a stable complex via histone fold domains (Goppelt et al 1996;Mermelstein et al 1996;Kamada et al 2001). In yeast, a homologous complex exists (Bur6/NC2a and Ydr1/NC2b), and it is required for cell growth (Gadbois et al 1997;Kim et al 1997;Prelich 1997). Several lines of evidence have suggested that NC2 functions as an inhibitor of pol II transcription.…”

Silencing of RNA Polymerases II and III-Dependent Transcription by the KRAB Protein Domain of KOX1, a Krüppel-Type Zinc Finger Factor

“…In this context it seems worth mentioning that the DR1/DRAP1 represser that is conserved from yeast to man has a similar activity spectrum as the KRAB domain described in here, in that it blocks transcription by RNA polymerases II and III but not polymerase I (White et a/., 1994;Kim et a/., 1997). DR1 binds to the TATA-binding protein TBP and blocks the interaction of TBP with polymerase II-and polymerase Ill-specific factors.…”

Regulation of Nuclear Import and Export of Negative Cofactor 2

“…Perhaps puzzling, whereas the downstream cluster of nucleoplasmin is not sufficient to target a protein into the nucleus (57,58), amino acids 1-10 of NC2␣ containing its cNLS ( 4 KKKK 7 ) functioned as an effective monopartite signal when fused to an EGFP-EGFP-GST reporter protein. Both NC2 genes are highly conserved in eukaryotes and are required for viability in yeast (1). However, the monopartite cNLS of neither NC2␣ nor NC2␤ is conserved in S. cerevisiae.…”

“…Both subunits are conserved in eukaryotes and essential for Saccharomyces cerevisae viability (1,2). NC2␣ and NC2␤ heterodimerize via histone-fold domains and associate with the promotor-bound TATA-binding protein (3,4).…”