ABSTRACT
A 64 x 32 liquid-crystal-on-silicon (LCOS) microdisplay with a frame buffer pixel architecture is described. The advantage of frame buffer pixel circuits is an increase in brightness and image contrast ratio. The increase in brightness is attributed to maximizing the overall image viewing time, allowing an image to be displayed at full contrast while the next image is loaded onto the pixels. The LCOS microdisplay employs a fast-switching optically compensated birefringence mode that operates at a 720-Hz frame frequency with a potentially high contrast ratio.
ABSTRACT
Transcription in eukaryotic cells requires the remodeling of chromatin and the assembly of functional preinitiation complexes (PICs), which contain the general transcription factors (GTFs), RNA polymerase II (Pol II), and coactivators. Genetic and biochemical studies have implicated the multisubunit Mediator coactivator complex (Med) as a critical component of the PIC, a direct target of activators, and a checkpoint for regulated gene expression during differentiation, development (reviewed in ), signaling, and oncogenesis. In this report, we show that a complex containing the activator GAL4-VP16, Med, and TFIID/TFIIA (DA) recruits pol II and the remaining GTFs to a model promoter in vitro. A preassembled DAMed complex bypasses the requirement for an activator. We also demonstrate that coordinated assembly of DAMed is essential to establishing a functional PIC. We conclude that the DAMed complex generates a platform that supports activated levels of PIC assembly and transcription.
Subject(s)
Gene Expression Regulation , Transcription Factor TFIIA/genetics , Transcription Factor TFIID/genetics , Transcription, Genetic/genetics , Autoradiography , DNA Polymerase II/genetics , Humans , Models, Genetic , Promoter Regions, Genetic/geneticsABSTRACT
HCF-1 functions as a coactivator for herpes simplex virus VP16 and a number of mammalian transcription factors. Mature HCF-1 is composed of two subunits generated by proteolytic cleavage of a larger precursor at six centrally-located HCF(PRO) repeats. The resulting N- and C-terminal subunits remain tightly associated via two complementary pairs of self-association domains: termed SAS1N-SAS1C and SAS2N-SAS2C. Additional HCF proteins have been identified in mammals (HCF-2) and Caenorhabditis elegans (CeHCF). Both contain well-conserved SAS1 domains but do not undergo proteolytic processing. Thus, the significance of the cleavage and self-association of HCF-1 remains enigmatic. Here, we describe the isolation of the Drosophila HCF homologue (dHCF) using a genetic screen based on conservation of the SAS1 interaction. The N-terminal beta-propeller domain of dHCF supports VP16-induced complex formation and is more similar to mammalian HCF-1 than other homologues. We show that full-length dHCF expressed in Drosophila cells undergoes proteolytic cleavage giving rise to tightly associated N- and C-terminal subunits. As with HCF-1, the SAS1N and SAS1C elements of dHCF are separated by a large central region, however, this sequence lacks obvious homology to the HCF(PRO) repeats required for HCF-1 cleavage. The conservation of HCF processing in insect cells argues that formation of separate N- and C-terminal subunits is important for HCF function.
Subject(s)
Drosophila/genetics , Peptide Hydrolases/metabolism , Proteins/genetics , Proteins/metabolism , Transcription Factors , Aging/physiology , Amino Acid Sequence/genetics , Animals , Cloning, Molecular , Conserved Sequence , DNA, Complementary/isolation & purification , Herpes Simplex Virus Protein Vmw65/physiology , Host Cell Factor C1 , Molecular Sequence Data , Protein Processing, Post-Translational , Protein Structure, Tertiary/physiology , Proteins/chemistry , Temperature , Transcription, GeneticABSTRACT
Activator-mediated transcription complex assembly on templates lacking chromatin requires the interaction of activators with two major coactivator complexes: TFIID and mediator. Here we employed immobilized template assays to correlate transcriptional activation with mediator and TFIID recruitment. In reactions reconstituted with purified proteins, we found that activator, TFIID, and mediator engage in reciprocal cooperative interactions to form a complex on promoter DNA. Preassembly of the coactivator complex accelerates the rate of transcription in a cell-free system depleted of TFIID and mediator. Our data argue that this coactivator complex is an intermediate in the assembly of an active transcription complex. Furthermore, the reciprocity of the interactions demonstrates that the complex could in principle be nucleated with either TFIID or mediator, implying that alternative pathways could be utilized to generate diversity in the way activators function in vivo.
