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C M Virbasius

Publications and source records attributed to C M Virbasius.

6 recordsLinked to original sources
PubMed2001

Promoter-specific activation defects by a novel yeast TBP mutant compromised for TFIIB interaction.

TFIIB is an RNA polymerase II general transcription factor (GTF) that has also been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, [1-11]). TFIIB enters the preinitiation complex (PIC) primarily through contact with the TATA box binding protein (TBP), an interaction mediated by three TBP residues [12-14]. To study the role of TFIIB in transcription activation in vivo, we randomly mutagenized these three residues in yeast TBP and screened for promoter-specific activation mutants. One mutant bearing a single conservative substitution, TBP-E186D, is the focus of this study. As expected, TBP-E186D binds normally to the TATA box but fails to support the entry of TFIIB into the PIC. Cells expressing TBP-E186D are viable but have a severe slow-growth phenotype. Whole-genome expression analysis indicates that transcription of 17% of yeast genes are compromised by this mutation. Chimeric promoter analysis indicates that the region of the gene that confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating sequence), which contains the activator binding sites. Most interestingly, other TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defective for interaction with TBP all manifest distinct and selective promoter-specific activation defects. Our results implicate the entry of TFIIB into the PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation.

Carrier Proteins↗
PubMed1999

A novel nuclear export activity in HIV-1 matrix protein required for viral replication.

An important aspect of the pathophysiology of human immunodeficiency virus type-1 (HIV-1) infection is the ability of the virus to replicate in non-dividing cells. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein (Pr55), bears a nuclear localization signal that promotes localization of the viral preintegration complex to the nucleus of non-dividing cells following virus entry. However, late during infection, MA, as part of Pr55, directs unspliced viral RNA to the plasma membrane, the site of virus assembly. How MA can mediate these two opposing targeting functions is not understood. Here we demonstrate that MA has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved Crm1p pathway and functions in both mammalian cells and yeast. A mutation that disrupts the MA nuclear export signal (MA-M4) mislocalizes Pr55 and genomic viral RNA to the nucleus, thereby severely impairing viral replication. Furthermore, we show that MA-M4 can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild-type MA. We conclude that the MA nuclear export signal is required to counteract the MA nuclear localization signal, thus ensuring the cytoplasmic availability of the components required for virion assembly.

Biological Transport↗
PubMed1999

A human nuclear-localized chaperone that regulates dimerization, DNA binding, and transcriptional activity of bZIP proteins.

We have identified and cloned a human nuclear protein that dramatically increases DNA binding of transcription factors that contain a basic region-leucine zipper (bZIP) DNA binding domain. We show that this bZIP enhancing factor (BEF) functions as a molecular chaperone. BEF stimulates DNA binding by recognizing the unfolded leucine zipper and promoting the folding of bZIP monomers to dimers; the elevated concentration of the bZIP dimer then drives the DNA binding reaction. Antisense experiments indicate that BEF is required for efficient transcriptional activation by bZIP proteins in vivo. Our results reveal protein folding in the nucleus as a step at which sequence-specific DNA binding proteins can be regulated.

Amino Acid Sequence↗
PubMed1996

Yeast TAF(II)90 is required for cell-cycle progression through G2/M but not for general transcription activation.

The RNA polymerase II general transcription factor TFIID is a multisubunit complex comprising TATA-box binding protein and associated factors (TAFIIs). In vitro experiments have suggested that TAFIIs are essential coactivators required for RNA polymerase II-directed transcription activation. Here, for the first time, we analyze systematically the in vivo function of a specific TAFII, yeast TAFII90 (yTAFII90). We show that functional inactivation of yTAFII90 by temperature-sensitive mutations or depletion leads to arrest at the G2/M phase of the cell cycle. Unexpectedly, in the absence of functional yTAFII90, a variety of endogenous yeast genes were all transcribed normally, including those driven by well-characterized activators. Taken together, our results indicate that yTAFII90 is not required for transcription activation in general, and reveal linkages between TAF function and cell-cycle progression.

Alleles↗
PubMed1996

Nuclear respiratory factors 1 and 2 utilize similar glutamine-containing clusters of hydrophobic residues to activate transcription.

Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) are ubiquitous transcription factors that have been implicated in the control of nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication. Recently, both factors have been found to be major transcriptional determinants for a subset of these genes that define a class of simple promoters involved in respiratory chain expression. Here, functional domains required for transactivation by NRF-1 have been defined. An atypical nuclear localization signal resides in a conserved amino-terminal region adjacent to the DNA binding domain and consists of functionally redundant clusters of basic residues. A second domain in the carboxy-terminal half of the molecule is necessary for transcriptional activation. The activation domains of both NRF-1 and NRF-2 were extensively characterized by both deletion and alanine substitution mutagenesis. The results show that these domains do not fall into known classes defined by a preponderance of amino acid residues, including glutamines, prolines, or isoleucines, as found in other eukaryotic activators. Rather, in both factors, a series of tandemly arranged clusters of hydrophobic amino acids were required for activation. Although all of the functional clusters contain glutamines, the glutamines differ from the hydrophobic residues in that they are inconsequential for activation. Unlike the NRF-2 domain, which contains its essential hydrophobic motifs within 40 residues, the NRF-1 domain spans about 40% of the molecule and appears to have a bipartite structure. The findings indicate that NRF-1 and NRF-2 utilize similar hydrophobic structural motifs for activating transcription.

Alanine↗