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PubMed1995

recO and recR mutations delay induction of the SOS response in Escherichia coli.

RecF, RecO and RecR, three of the important proteins of the RecF pathway of recombination, are also needed for repair of DNA damage due to UV irradiation. recF mutants are not proficient in cleaving LexA repressor in vivo following DNA damage: therefore they show a delay of induction of the SOS response. In this communication, by measuring the in vivo levels of LexA repressor using anti-LexA antibodies, we show that recO and recR mutant strains are also not proficient in LexA cleavage reactions. In addition, we show that recO and recR mutations delay induction of beta-galactosidase activity expressed from a lexA-regulated promoter following exposure of cells to UV, thus further supporting the idea that recF, recO and recR gene products are needed for induction of the SOS response.

Bacterial Proteins↗
PubMed1989

The LexA protein does not bind specifically to the two SOS box-like sequences immediately 5' to the phr gene.

There are two SOS box-like sequences located at the regions -161 to -142 and -69 to -50 with regard to the initiation codon of phr. Ihara et al. (1987) constructed a phr'-'lacZ fusion plasmid in which these sequences and the amino terminal end of the phr gene was fused to lacZ and therefore lacZ was under the regulatory control of phr promoter-operator. The authors found that in cells carrying this plasmid beta-galactosidase was inducible by UV and UV-mimetic agents and concluded that phr was controlled by the LexA repressor. We wished to confirm these results by theoretical analysis of the SOS-like sequences by the method developed by Berg (1987) as well as by measuring the binding of LexA protein to the putative SOS boxes by a novel gel retardation assay. Both theoretical analysis and experimental results indicate that the putative SOS boxes immediately 5' to phr have no specific affinity for LexA protein.

Bacterial Proteins↗
PubMed1991

A yeast transcription assay defines distinct rel and dorsal DNA recognition sequences.

Recent data have demonstrated that vRel, cRel, Dorsal, and NF-kappa B are members of a larger family of DNA-binding regulatory proteins. Rel proteins interact to form homo- and heterodimers that recognize specific sites on DNA, and it is likely that such protein-protein and protein-DNA interactions contribute to proper regulation of target gene expression by these proteins. Here we describe the use of a yeast transcription activation assay to study binding of three Rel family proteins to their native binding sites. These results show that the vRel and cRel proteins recognize two known NF-kappa B binding sites; the Dorsal protein does not recognize NF-kappa B sites, but does recognize related sites upstream of the Drosophila zerknüllt gene. Our experiments demonstrate that the members of this protein family recognize similar, but not identical, sites in the promoters of target genes, and we are able to identify a particular nucleotide that is apparently involved in the DNA-protein interaction. We exploit the properties of LexA fusion proteins to study the dimerization and DNA-contacting domains of cRel. Our results suggest that the cRel protein forms homodimers and that dimer formation may be necessary for cRel to bind DNA. Finally, our results show that transcription activation by these proteins is cooperative; such cooperativity may be important for correct temporal and spatial regulation of target gene expression.

Animals↗
PubMed1997

Use of a two-hybrid system to identify mutations in Max that confer increased affinity for Myc.

A yeast two-hybrid system was used to identify mutants of Max that exhibit an increased affinity for Myc. Truncated forms of the Max helix-loop-helix/leucine zipper motif (HLH/Zip) were first expressed in a two- hybrid system in which the bait protein was the HLH/Zip motif of Myc. Deletion of amino acids both amino-terminal and carboxy-terminal to the leucine zipper of Max reduced Myc/Max heterodimer formation as evidenced by a 160-fold reduction in the expression of the lacZ gene. A library of partially randomized sequences encoding this minimal leucine zipper of Max was then screened using the two-hybrid system. Mutant forms of the Max leucine zipper were identified whose affinities for Myc, as measured by beta-galactosidase activity in yeast lysates, were from 8- to 200-fold greater than the wild-type Max zipper. These Max mutants were shown to interact specifically with Myc and not with wild-type Max. Of 29 mutants analyzed, all had a unique amino acid sequence. This result illustrates the value of a genetic screen in the identification of a collection of mutant forms of the Max leucine zipper whose structures would not have been predicted based on principles of structure-based design.

Bacterial Proteins↗
PubMed2005

Expression of one-hybrid fusions with Staphylococcus aureus lexA in Candida albicans confirms that Nrg1 is a transcriptional repressor and that Gcn4 is a transcriptional activator.

In the pathogenic fungus, Candida albicans, Nrg1 down-regulates the expression of morphogenetic genes and is presumed to act as a transcriptional repressor. In contrast, Gcn4 up-regulates amino acid biosynthetic genes and is presumed to be a transcriptional activator. However, these presumptions remain to be tested directly. A classic approach has been to use a one-hybrid assay that exploits the Escherichia coli lexA protein fusions. However in C. albicans, the alternate decoding of CUG as serine prevents the expression of heterologous genes such as lexA, which contain numerous CUG codons. Therefore, we have developed a one-hybrid system, based on the Staphylococcus aureus lexA gene, as a tool for one-hybrid analyses of transcription factors in C. albicans. Using this one-hybrid system we have confirmed directly the positive and negative transcriptional activities of Nrg1 and Gcn4 in C. albicans.

Bacterial Proteins↗
PubMed1991

Identification of the DNA binding site for NGFI-B by genetic selection in yeast.

An in vivo selection system for isolating targets of DNA binding proteins in yeast was developed and used to identify the DNA binding site for the NGFI-B protein, a member of the steroid-thyroid hormone receptor superfamily. The feasibility of the technique was verified by selecting DNA fragments that contained binding sites for GCN4, a well-characterized yeast transcriptional activator. The DNA binding domain of NGFI-B, expressed as part of a LexA-NGFI-B-GAL4 chimeric activator, was then used to isolate a rat genomic DNA fragment that contained an NGFI-B binding site. The NGFI-B response element (NBRE) is similar to but functionally distinct from elements recognized by the estrogen and thyroid hormone receptors and the hormone receptor-like proteins COUP-TF, CF1, and H-2RIIBP. Cotransfection experiments in mammalian cells demonstrated that NGFI-B can activate transcription from the NBRE with or without its putative ligand binding domain.

Animals↗
PubMed2000

A genetic system for detection of protein nuclear import and export.

We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.

Amino Acid Sequence↗
PubMed2000

LexA chimeras reveal the function of Drosophila Fos as a context-dependent transcriptional activator.

The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays. Here, we applied it to assay the transactivation potential of proteins in transgenic Drosophila embryos. We found that a chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaved as a potent synthetic activator in all embryonic tissues. In contrast, a LexA chimera containing Drosophila Fos (Dfos) required an unexpected degree of context to function as a transcriptional activator. We provide evidence to suggest that this context is provided by Djun and Mad (a Drosophila Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, our data suggest that context-dependence of transcription factors may be more prevalent than previously thought.

Animals↗
PubMed1997

Hir1p and Hir2p function as transcriptional corepressors to regulate histone gene transcription in the Saccharomyces cerevisiae cell cycle.

The HIR/HPC (histone regulation/histone periodic control) negative regulators play important roles in the transcription of six of the eight core histone genes during the Saccharomyces cerevisiae cell cycle. The phenotypes of hir1 and hir2 mutants suggested that the wild-type HIR1 and HIR2 genes encode transcriptional repressors that function in the absence of direct DNA binding. When Hir1p and Hir2p were artificially tethered to yeast promoters, each protein repressed transcription, suggesting that they represent a new class of transcriptional corepressors. The two proteins might function as a complex in vivo: Hir2p required both Hir1p and another Hir protein, Hir3p, to repress transcription when it was tethered to an HTA1-lacZ reporter gene, and Hir1p and Hir2p could be coimmunoprecipitated from yeast cell extracts. Tethered Hir1p also directed the periodic transcription of the HTA1 gene and repressed HTA1 transcription in response to two cell cycle regulatory signals. Thus, it represents the first example of a transcriptional corepressor with a direct role in cell cycle-regulated transcription.

Bacterial Proteins↗
PubMed1996

I-mf, a novel myogenic repressor, interacts with members of the MyoD family.

During embryogenesis, cells from the ventral and dorsal parts of the somites give rise to sclerotome and dermomyotome, respectively. Dermomyotome contains skeletal muscle precursors that are determined by the MyoD family of myogenic factors. We have isolated a novel myogenic repressor, I-mf (Inhibitor of MyoD family), which is highly expressed in the sclerotome. In contrast, MyoD family members are concentrated in the dermomyotome. We demonstrate that I-mf inhibits the transactivation activity of the MyoD family and represses myogenesis. I-mf associates with MyoD family members and retains them in the cytoplasm by masking their nuclear localization signals. I-mf can also interfere with the DNA binding activity of MyoD family members. We postulate that I-mf plays an important role in the patterning of the somite early in development.

3T3 Cells↗
PubMed2003

Collaborative competition mechanism for gene activation in vivo.

The mechanism by which gene regulatory proteins gain access to their DNA target sites is not known. In vitro, binding is inherently cooperative between arbitrary DNA binding proteins whose target sites are located within the same nucleosome. We refer to such competition-based cooperativity as collaborative competition. Here we show that arbitrarily chosen foreign DNA binding proteins, LexA and Tet repressor, cooperate with an adjacently binding endogenous activator protein, Gcn4, to coactivate expression of chromosomal reporter genes in Saccharomyces cerevisiae. Coactivation requires that the cooperating target sites be within a nucleosome-length distance; it leads to increased occupancy by Gcn4 at its binding site; and it requires both Gcn5 and Swi/Snf which, at an endogenous Gcn4-dependent promoter, act subsequent to Gcn4 binding. These results imply that collaborative competition contributes to gene regulation in vivo. They further imply that, even in the presence of the cell's full wild-type complement of chromatin remodeling factors, competition of regulatory proteins with histone octamer for access to regulatory target sites remains a quantitative determinant of gene expression levels. We speculate that initial target site recognition and binding may occur via spontaneous nucleosomal site exposure, with remodeling factor action required downstream to lock in higher levels of regulatory protein occupancy.

Adenosine Triphosphate↗
PubMed1997

Complementation of a yeast delta pkc1 mutant by the Arabidopsis proteinANT.

The Saccharomyces cerevisiae protein kinase C homologue, PKC1, is involved in maintenance of cell integrity during polarized growth. We have used a mutant complementation approach to investigate related signal transduction pathways in higher plants. Here we report the isolation of a cDNA from Arabidopsis thaliana which partially suppresses the lytic defect of a delta pkc1 yeast strain. The encoded protein, ANT, belongs to the AP2-related gene family and is essential for ovule development. Expression in yeast of a LexA-ANT fusion protein activates transcription of a reporter gene from promoters containing lexA operators. Our results support the idea that ANT acts as transcriptional activator in planta.

Arabidopsis↗
PubMed2000

Genetic analysis of the role of Pol II holoenzyme components in repression by the Cyc8-Tup1 corepressor in yeast.

The Cyc8-Tup1 corepressor complex is targeted to promoters by pathway-specific DNA-binding repressors, thereby inhibiting the transcription of specific classes of genes. Genetic screens have identified mutations in a variety of Pol II holoenzyme components (Srb8, Srb9, Srb10, Srb11, Sin4, Rgr1, Rox3, and Hrs1) and in the N-terminal tails of histones H3 and H4 that weaken repression by Cyc8-Tup1. Here, we analyze the effect of individual and multiple mutations in many of these components on transcriptional repression of natural promoters that are regulated by Cyc8-Tup1. In all cases tested, individual mutations have a very modest effect on SUC2 RNA levels and no detectable effect on levels of ANB1, MFA2, and RNR2. Furthermore, multiple mutations within the Srb components, between Srbs and Sin4, and between Srbs and histone tails affect Cyc8-Tup1 repression to the same modest extent as the individual mutations. These results argue that the weak effects of the various mutations on repression by Cyc8-Tup1 are not due to redundancy among components of the Pol II machinery, and they argue against a simple redundancy between the holoenzyme and chromatin pathways. In addition, phenotypic analysis indicates that, although Srbs8-11 are indistinguishable with respect to Cyc8-Tup1 repression, the individual Srbs are functionally distinct in other respects. Genetic interactions among srb mutations imply that a balance between the activities of Srb8 + Srb10 and Srb11 is important for normal cell growth.

Bacterial Proteins↗
PubMed1999

Interaction between F plasmid partition proteins SopA and SopB.

Two different yeast two hybrid systems were used to examine interaction between the partition proteins SopA and SopB of F plasmid as well as their self association. In one system, the yeast Gal4 protein DNA binding domain (Gal4-BD) is fused to the N-terminus of the bait protein, and the Gal4 activation domain (Gal4-AD) is fused to the N-terminus of the target protein (1). In the other system, the target hybrid remains unchanged but E. coli LexA protein (LexA) is fused to the C-terminus of the bait protein (2). It is found that C-terminus part of SopB is involved in interaction with itself, as an N-terminal truncation of SopB, SopB-(120-323) remains capable of self association. For interaction between SopA, deletion of the N-terminal part weakens but does not abolish the interaction. Interaction between SopB and SopA protein was also detected, but only by the use of the second system. Full length SopB [SopB-(1-323)] or SopB-(1-180) lacking the C-terminal region beyond amino acid 180 can interact with full-length SopA-(1-383) protein.

Bacterial Proteins↗
PubMed1992

Activating regions of yeast transcription factors must have both acidic and hydrophobic amino acids.

Two similarities among transcriptional activating regions of many eukaryotic transcription factors, like those from GAL4, GCN4, and VP16, are that they have a net negative charge, and that many of them can potentially form amphipathic alpha-helices with acidic amino acids on the hydrophilic face. Based on these similarities, E. Giniger and M. Ptashne previously designed a short peptide (AH) which is predicted to have the potential to form a negatively charged amphipathic alpha-helix; AH was able to mediate transcription activation in yeast when it was attached to the DNA binding and dimerization portion of GAL4 [GAL4(1-147)]. This paper describes screening of a pool of AH derivatives containing randomized amino acids fused to GAL4(1-147) and to an analogous region of LexA [LexA(1-87)] in yeast strains. Results suggest that both acidic and hydrophobic amino acids are critical features of activating regions--these results are consistent with the model that activating regions often form amphipathic alpha-helices. This work is novel because hydrophobic amino acids are also shown to be important in activating regions of yeast transcription factors.

Amino Acid Sequence↗
PubMed1994

Applications of interaction traps/two-hybrid systems to biotechnology research.

Two-hybrid methods provide a simple and sensitive means to detect the interaction between two proteins in living cells. Their use has resulted in the isolation of new proteins and has facilitated characterization of particular protein-protein interactions. These techniques have already resulted in the identification of important targets for pharmaceutical intervention, and it is likely that their extension in coming years will facilitate the development of new drugs.

Bacterial Proteins↗