cDNA expression cloning and characterization of phosphorylation dependent protein interactors using the yeast tribrid system.
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Amino Acid Sequence↗
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Results for “LexA Repressor Protein”
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The split-hybrid system. Uncoding multiprotein networks and defining mutations that affect protein interactions.
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Bacterial Proteins↗
Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein.
The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.
ATP-Binding Cassette Transporters↗
Variability in intrinsic promoter strength underlies the temporal hierarchy of the Caulobacter SOS response induction.
Bacteria encode for gene regulatory networks crucial for sensing and repairing DNA damage. Upon exposure to genotoxic stress, these transcriptional networks are induced in a temporally structured manner. A case in point is of the highly conserved SOS response that is regulated by the LexA repressor. Studies have proposed that affinity of LexA towards promoters of SOS response genes is the primary determinant of its expression dynamics. Here, we describe an additional level of regulation beyond LexA box properties that modulates the SOS response gene expression pattern. Using transcriptomic analyses, we reveal a distinct temporal hierarchy in the induction of SOS-regulated genes in Caulobacter crescentus. We observe that LexA box properties are insufficient in predicting the temporal hierarchy of these genes. Instead, we find that intrinsic promoter strength underlies the order of gene activation, with differential sigma factor association as one of the factors modulating gene expression timing. Our findings highlight a novel regulatory layer in SOS dynamics and underscore the importance of promoter properties in shaping bacterial stress responses.
Promoter Regions, Genetic↗
TUP1 utilizes histone H3/H2B-specific HDA1 deacetylase to repress gene activity in yeast.
TUP1 is recruited to and represses genes that regulate mating, glucose and oxygen use, stress response, and DNA damage. It is shown here that disruption of either TUP1 or histone deacetylase HDA1 causes histone H3/H2B--specific hyperacetylation next to the TUP1 binding site at the stress-responsive ENA1 promoter. It is also shown that TUP1 interacts with HDA1 in vitro. These data indicate that TUP1 mediates localized histone deacetylation through HDA1. Interestingly, RPD3 deacetylates the ENA1 coding region, and both deacetylases contribute to ENA1 repression. However, epistasis analysis argues that only HDA1 and TUP1 are likely to function in the same pathway. These data define gene and histone targets of HDA1 and illustrate the role of histone deacetylation in TUP1 repression.
ATP-Binding Cassette Transporters↗
Improved model of a LexA repressor dimer bound to recA operator.
A complete three dimensional model for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor was proposed from our laboratory when no crystal structure of LexA was available. Subsequently, the crystal structures of four LexA mutants Delta(1-67) S119A, S119A, G85D and Delta(1-67) quadruple mutant in the absence of operator were reported. It is examined in this paper to what extent our previous model was correct and how, using the crystal structure of the operator-free LexA dimer we can predict an improved model of LexA dimer bound to recA operator. In our improved model, the C-domain dimerization observed repeatedly in the mutant operator-free crystals is retained but the relative orientation between the two domains within a LexA molecule changes. The crystal structure of wild type LexA with or without the recA operator cannot be solved as it autocleaves itself. We argue that the 'cleavable' cleavage site region found in the crystal structures is actually the more relevant form of the region in wild-type LexA since it agrees with the value of the pre-exponential Arrhenius factor for its autocleavage, absence of various types of trans-cleavages, difficulty in modifying the catalytic serine by diisopropyl flourophosphate and lack of cleavage at Arg 81 by trypsin; hence the concept of a 'conformational switch' inferred from the crystal structures is meaningless.
Amino Acid Sequence↗
Model of a LexA repressor dimer bound to recA operator.
A complete three dimensional model (RCSB000408; PDB code 1qaa) for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor is proposed. A model of interaction of the N-terminal operator binding domain 1-72 with the operator was available. We have modelled residues 106-202 of LexA on the basis of the crystal structure of a homologous protein, UmuD'. Residues 70-105 have been modelled by us, residues 70-77 comprising the real hinge, followed by a beta-strand and an alpha-helix, both interacting with the rest of the C-domain. The preexponential Arrhenius factor for the LexA autocleavage is shown to be approximately 10(9) s(-1) at 298K whereas the exponential factor is approximately 2 x 10(-12), demanding that the autocleavage site is quite close to the catalytic site but reaction is slow due to an activation energy barrier. We propose that in the operator bound form, Ala 84- Gly 85 is about 7-10A from the catalytic groups, but the reaction does not occur as the geometry is not suitable for a nucleophilic attack from Ser 119 Ogamma, since Pro 87 is held in the cis conformation. When pH is elevated or under the action of activated RecA, cleavage may occur following a cis --> trans isomerization at Pro 87 and/or a rotation of the region beta9-beta10 about beta7-beta8 following the disruption of two hydrogen bonds. We show that the C-C interaction comprises the approach of two negatively charged surfaces neutralized by sodium ions, the C-domains of the monomers making a new beta barrel at the interface burying 710A2 of total surface area of each monomer.
Amino Acid Sequence↗
The SOS system of Escherichia coli in the regulation of bacteriophage lambda development.
A sequence homologous to the known SOS boxes is found in the Po promoter of phage lambda. It is suggested that the sequence found is a binding site for the LexA repressor. The mechanism of the LexA part in regulation of lambda development is discussed. It is based on the competitive transcription of the RNA encoding CII protein and the short OOP-RNA transcribed from the Po promoter.
Amino Acid Sequence↗
IRE1- and HAC1-independent transcriptional regulation in the unfolded protein response of yeast.
The unfolded protein response (UPR) is a signalling pathway leading to transcriptional activation of genes that protect cells from accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER). In yeast, the only known ER stress signalling pathway originates at the type I transmembrane protein kinase/endoribonuclease Ire1p. Ire1p regulates synthesis of the basic leucine-zipper (bZIP)-containing transcription factor Hac1p by controlling splicing of HAC1 mRNA. Only spliced HAC1 mRNA (HAC1i) is translated, and Hac1ip activates transcription of genes that contain a conserved UPR element (UPRE) in their promoters. Here, we demonstrate that in addition to this well-understood ER stress signalling pathway, a second, IRE1, HAC1 and UPRE-independent mechanism for transcriptional activation upon ER stress, exists in yeast. A genetic screen identified recessive SIN4 alleles as suppressors of a defective UPR in ire1 Delta strains. Elevation of basal transcription in sin4 strains or by tethering the RNA polymerase II holoenzyme with LexAp-holoenzyme component fusion proteins to a promoter allowed for activation of the promoter by ER stress in an IRE1, HAC1 and UPRE-independent manner. We propose that this novel second ER-to-nucleus signal transduction pathway culminates in core promoter activation (CPA) through stimulation of RNA polymerase II holoenzyme activity. Core promoter activation was observed upon diverse cellular stresses, suggesting it represents a primordial stress-induced gene activation mechanism.
Alleles↗
Identification of protein-protein interactions of the major sperm protein (MSP) of Caenorhabditis elegans.
In nematodes, sperm are amoeboid cells that crawl via an extended pseudopod. Unlike those in other crawling cells, this pseudopod contains little or no actin; instead, it utilizes the major sperm protein (MSP). In vivo and in vitro studies of Ascaris suum MSP have demonstrated that motility occurs via the regulated assembly and disassembly of MSP filaments. Filaments composed of MSP dimers are thought to provide the motive force. We have employed the yeast two-hybrid system to investigate MSP-MSP interactions and provide insights into the process of MSP filament formation. Fusions of the Caenorhabditis elegans msp-142 gene to both the lexA DNA binding domain (LEXA-MSP) and a transcriptional activation domain (AD-MSP) interact to drive expression of a lacZ reporter construct. A library of AD-MSP mutants was generated via mutagenic PCR and screened for clones that fail to interact with LEXA-MSP. Single missense mutations were identified and mapped to the crystal structure of A. suum MSP. Two classes of mutations predicted from the structure were recovered: changes in residues critical for the overall fold of the protein, and changes in residues in the dimerization interface. Multiple additional mutations were obtained in the two carboxy-terminal beta strands, a region not predicted to be involved in protein folding or dimer formation. Size fractionation of bacterially expressed MSPs indicates that mutations in this region do not abolish dimer formation. A number of compensating mutations that restore the interaction also map to this region. The data suggest that the carboxy-terminal beta strands are directly involved in interactions required for MSP filament assembly.
Amino Acid Sequence↗
LexA-binding sequences in Gram-positive and cyanobacteria are closely related.
The lexA gene of the cyanobacterium Anabaena sp. strain PCC7120 has been cloned by PCR amplification with primers designed after TBLASTN analysis of its genome sequence using the Escherichia coli LexA sequence as a probe. After over-expression in E. coli and subsequent purification, footprinting experiments demonstrated that the Anabaena LexA protein binds to the sequence TAGTACTAATGTTCTA, which is found upstream of its own coding gene. Directed mutagenesis and sequence comparison of promoters of other Anabaena genes, as well as those of several cyanobacteria, allowed us to define the motif RGTACNNNDGTWCB as the LexA box in this bacterial phylum. Substitution of a single nucleotide in this motif present in the Anabena lexA promoter is sufficient to enable it to bind the Bacillus subtilis LexA protein. These data indicate that Cyanobacteria and Gram-positive bacteria are phylogenetically closely related.
Amino Acid Motifs↗
Backbone chemical shift assignments of the LexA catalytic domain in its active conformation.
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Bacterial Proteins↗
Interaction of p85 subunit of PI 3-kinase with insulin and IGF-1 receptors analysed by using the two-hybrid system.
Interaction of the p85 subunit of PI 3-kinase with the insulin receptor (IR) and the IGF-1 receptor (IGF-1R) was investigated using the two-hybrid system by assessing for his3 and lacZ activation in S. cerevisiae. The experiments were performed with the cytoplasmic beta domain (wild type or mutated) of IR and IGF-1R and p85 or its subdomains (N + C-SH2, N-SH2, C-SH2, SH3 + N-SH2). The results of his3 activation indicated that p85, N + C-SH2 and C-SH2 interact with both IR beta and IGF-1R beta, whereas N-SH2 and SH3 + N-SH2 interact only with IR beta. Interaction of p85 and N+C-SH2 with IR beta (delta C-43) or IGF-1R beta(delta C-43) in which the C-terminal 43 amino acids (including the YXXM motif) were deleted, persisted. The internal binding site thus revealed was not altered by further mutating Y960/F for IR or Y950/F for IGF-1R. Activation of lacZ upon interaction of p85 with IR beta(delta C-43) was 4-fold less as compared to IR beta. This activation with p85 and IGF-1R beta was 4-fold less as compared to IR beta and was somewhat increased (2-fold) for IGF-1R beta (delta C-43). Thus, the C-terminal domain in IGF-1R appears to exert a negative control on binding of p85 thereby providing a possible regulatory mechanism for direct activation of the PI 3-kinase pathway.
Amino Acid Sequence↗
Mechanism of transcriptional silencing in yeast.
Transcriptional silencing is a phenomenon in which the transcription of a gene by RNA polymerase II or III is repressed or not, dependent only on the gene's chromosomal location. Two prevailing models exist for silencing: (1) steric hindrance in silenced chromatin inhibits the binding of upstream activator proteins or polymerase or (2) silencing primarily blocks steps downstream of transcription preinitiation complex formation. Here, we test these models quantitatively for the case of SIR2-dependent silencing in budding yeast, using foreign and endogenous reporter proteins, at transgenic and endogenous loci. Our results contradict both models and show instead that transcriptional silencing at several URA3 transgenes, and at the naturally silenced endogenous HMRa and HMLalpha mating type genes, acts downstream of gene activator protein binding to strongly reduce the occupancy of TFIIB, RNA polymerase II, and TFIIE at the silenced promoters.
Bacterial Proteins↗
Using the yeast three-hybrid system to detect and analyze RNA-protein interactions.
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Bacterial Proteins↗
Activator-mediated recruitment of the RNA polymerase II machinery is the predominant mechanism for transcriptional activation in yeast.
Eukaryotic transcriptional activators bind to enhancer elements and stimulate the RNA polymerase II (pol II) machinery via functionally autonomous activation domains. In yeast cells, the normal requirement for an activation domain can be bypassed by artificially connecting an enhancer-bound protein to a component of the pol II machinery. This observation suggests, but does not necessarily indicate, that the physiological role of activation domains is to recruit the pol II apparatus to promoters. Here, we show that transcriptional stimulation does not occur when the activation domain is physically disconnected from the enhancer-bound protein and transferred to components of the pol II machinery. The observation that autonomous activation domains are functional when connected to enhancer-bound proteins but not to components of the pol II machinery strongly argues that recruitment is the predominant mechanism for transcriptional activation in yeast.
Bacterial Proteins↗
Induced protein dimerization in vivo through covalent labeling.
We present here a novel approach to induce protein dimerization in living cells through covalent labeling of fusion proteins with ligands which are interacting with other proteins. The covalent labeling is based on the reaction of the DNA repair protein O6-alkylguanine-DNA alkyltransferase with O6-benzylguanine coupled to an appropriate ligand. Using methotrexate as a ligand, it is shown that the approach can be used to control transcription in the yeast Saccharomyces cerevisiae. The specificity of the labeling reaction and its irreversible nature should allow the approach to become a valuable tool to study and control protein dimerization in vivo.
Bacterial Proteins↗
Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers.
The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.
Alzheimer Disease↗