A Negative Cofactor Containing Dr1/p19 Modulates Transcription with TFIIA in a Promoter-specific Fashion

Abstract

An activity that modulated the relative levels of transcription from the adenovirus major late promoter (MLP), and the immunoglobulin heavy chain promoter () was purified as a 90-kDa factor. This factor is suggested to be a heterotetramer of two subunits: a 20-kDa polypeptide identical to the previously described Dr1/ p19 and a novel 30-kDa polypeptide. The Dr1/p19 protein has been characterized as a repressor of transcription, and the 30-kDa protein is related to a recently identified yeast gene proposed to encode a repressor of transcription. The 90-kDa factor forms a complex with TATAbinding protein on DNA and at high concentrations of both factors protects over a 150-base pair region around the promoter from DNase I cleavage. The conformation of this complex as assayed by footprinting analysis is altered by the transcription factor TFIIA on the MLP but not on the promoter. Similarly, TFIIA reverses the repression of transcription by the 90-kDa factor on the MLP but not on the promoter. Thus, the interactions of TATA-binding protein, TFIIA, and the 90-kDa factor are promoter-specific. Transcription reactions in vitrowere developed to analyze the synthesis of pre-mRNA from promoter DNA. Reconstitution of transcription with chromatographic fractions has allowed purification of general transcription factors and isolation of the genes encoding the corresponding polypeptides (for review see Refs. 1-3). Also identified were activities dispensable for the basal levels of transcription that affected the absolute amounts of product RNA. Initially, activities that suppressed transcription in vitro were largely ignored, but as addition of these activities was shown often to increase the relative response to sequence-specific transcriptional activators, interest in their identity and mechanism grew (4 -6). These are collectively referred to as negative cofactors of transcription and are distinct, at least operationally, from silencer element-dependent proteins in that they repress a wide variety of promoters.Several lines of evidence suggest that repression of transcription is a general and important aspect of transcriptional regulation. Both genetic and biochemical experiments suggest that chromatin structure represses transcription (for review see Refs. 7 and 8). Furthermore, studies of SWI/SNF complexes show that controlling the accessibility of chromatin bound promoters to transcription factors is one of the regulatory steps of transcription (9 -12). Genetic screening for yeast mutants with elevated basal transcription and for suppressors of mutations to specific upstream activating sequences has led to the identification of several so-called global repressors of polymerase II (pol II) 1 transcription (13-17). Mutations to many of them have pleiotropic effects, often elevating the level of transcription from multiple promoters. The SRB family of proteins, found as suppressors of C-terminal domain truncation mutations of pol II, are implicated as important components of transcription initiation in vivo (for review see...

Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2 in vivo

“…These results extend the known negative regulatory effects of NC2 on core RNA polymerase II in vitro (16,17,20,(42)(43)(44)(47)(48)(49)(50) and confirm that this regulator can inhibit transcription by RNA polymerase II in vivo.…”

“…1b). A search of the GenBank database (June 7, 1996) revealed that the predicted protein has 39% identity over 99 aa to the NC2␣ (DRAP1) subunit of human NC2 (Dr1⅐DRAP1), which binds to TBP and represses transcription in vitro (16,17,20,(42)(43)(44)(47)(48)(49)(50). The gene encoding the putative yeast NC2␣ protein was named NCB1.…”

“…Several lines of evidence argue that NC2 acts globally as a repressor of most, if not all, class II genes. NC2 can repress transcription in vitro from a wide variety of mammalian, viral, and yeast promoters (16,20,(42)(43)(44)50). The NC2 suppressor mutation that compensates for reduced class II gene transcription in vivo due to loss of SRB4 also compensates for the global defect due to loss of SRB6 (data not shown).…”

“…The negative regulators that have been described include proteins purified for their ability to inhibit transcription in vitro (15-21) and genes identified because their products repress transcription from a subset of class II genes in vivo (21-29). For example, the human proteins NC1 (15, 16), NC2 or Dr1⅐DRAP1 (16,17,20), and DNA topoisomerase I (18, 19) repress basal transcription in vitro. The products of the yeast genes MOT1 (21-24), NOT1-4 (25-27), and SIN4 (28-29) negatively regulate at least a subset of yeast genes in vivo.…”

Purification and Enzymic Properties of Mot1 ATPase, a Regulator of Basal Transcription in the Yeast Saccharomyces cerevisiae

“…Extracts were prepared immediately thereafter by vigorous vortex mixing with glass beads and, after clarification, were loaded in parallel onto Ni 2ϩ -iminodiacetate-agarose columns. The columns were washed with the same buffer, then eluted using the same buffer containing increasing concentrations of imidazole (20,40,60, and 500 mM), and then stripped with Binding Buffer ϩ 100 mM EDTA. Proteins present in the eluted fractions were analyzed by SDS-PAGE and immunoblotting with an appropriate antibody.…”