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PubMed1996

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↗
PubMed1995

Identification of the potential active site of the signal peptidase SipS of Bacillus subtilis. Structural and functional similarities with LexA-like proteases.

Signal peptidases remove signal peptides from secretory proteins. By comparing the type I signal peptidase, SipS, of Bacillus subtilis with signal peptidases from prokaryotes, mitochondria, and the endoplasmic reticular membrane, patterns of conserved amino acids were discovered. The conserved residues of SipS were altered by site-directed mutagenesis. Replacement of methionine 44 by alanine yielded an enzyme with increased activity. Two residues (aspartic acid 146 and arginine 84) appeared to be conformational determinants; three other residues (serine 43, lysine 83, and aspartic acid 153) were critical for activity. Comparison of SipS with other proteases requiring serine, lysine, or aspartic acid residues in catalysis revealed sequence similarity between the region of SipS around serine 43 and lysine 83 and the active-site region of LexA-like proteases. Furthermore, self-cleavage sites of LexA-like proteases closely resembled signal peptidase cleavage sites. Together with the finding that serine and lysine residues are critical for activity of the signal peptidase of Escherichia coli (Tschantz, W.R., Sung, M., Delgado-Partin, V.M., and Dalbey, R.E. (1993) J. Biol. Chem. 268, 27349-27354), our data indicate that type I signal peptidases and LexA-like proteases are structurally and functionally related serine proteases. A model envisaging a catalytic serine-lysine dyad in prokaryotic type I signal peptidases is proposed to accommodate our observations.

Amino Acid Sequence↗
PubMed2006

A novel yeast-based reporter assay system for the sensitive detection of genotoxic agents mediated by a DNA damage-inducible LexA-GAL4 protein.

Yeast-based genotoxicity testing systems can sensitively detect DNA damaging agents in the environment. We have developed a novel "indirect" reporter assay system based on a recombinant yeast containing both a sensor and a reporter plasmid. The sensor plasmid contains a gene encoding the artificial transcription factor of the Escherichia coli LexA DNA binding domain fused to the transcriptional activation domain of yeast Gal4p, which is regulated by the DNA damage-inducible RNR2 promoter. The reporter plasmid contains the E. coli lacZ gene with the LexA binding site in the 5'-upstream region, allowing transcriptional activation by the induced LexA-GAL4 protein. The activity of DNA damage-dependent beta-galactosidase (beta-gal) in the "indirect" reporter assay system was compared with that of a current yeast-based "direct" reporter system. The "indirect" system exhibited 1.5- to 5-fold greater beta-gal activity upon induction by alkylating agents or camptothecin. To increase the sensitivity of the new reporter system further, several deletion yeast strains were tested, and enhanced induction of reporter activity was observed in DNA repair-deficient mag1Delta cells. The "indirect" 96-well microtiter plate assay system is a potentially inexpensive and sensitive method for detecting genotoxic activities in a wide range of compounds, and in polluted environmental samples.

Bacterial Proteins↗
PubMed1988

Yeast HAP2 and HAP3: transcriptional activators in a heteromeric complex.

Transcription of the yeast C upsilon C1 gene (iso-1-cytochrome c) is regulated in part by the upstream activation site UAS2. Activity of UAS2 requires both the HAP2 and HAP3 activators, which bind to UAS2 in an interdependent manner. To distinguish whether these factors bound to UAS2 cooperatively or formed a complex in the absence of DNA, HAP2 and HAP3 were tagged by gene fusion to LexA and beta-galactosidase, respectively, and purified through four chromatographic steps. The copurification of LexA-HAP2, HAP3 beta-galactosidase, and UAS2 binding activity shows that HAP2 and HAP3 associate in the absence of DNA to form a multisubunit activation complex.

Bacterial Proteins↗
PubMed2006

Fine-structure analysis of ribosomal protein gene transcription.

The ribosomal protein genes of Saccharomyces cerevisiae, responsible for nearly 40% of the polymerase II transcription initiation events, are characterized by the constitutive tight binding of the transcription factor Rap1. Rap1 binds at many places in the yeast genome, including glycolytic enzyme genes, the silent MAT loci, and telomeres, its specificity arising from specific cofactors recruited at the appropriate genes. At the ribosomal protein genes two such cofactors have recently been identified as Fhl1 and Ifh1. We have now characterized the interaction of these factors at a bidirectional ribosomal protein promoter by replacing the Rap1 sites with LexA operator sites. LexA-Gal4(AD) drives active transcription at this modified promoter, although not always at the correct initiation site. Tethering Rap1 to the promoter neither drives transcription nor recruits Fhl1 or Ifh1, showing that Rap1 function requires direct DNA binding. Tethering Fhl1 also fails to activate transcription, even though it does recruit Ifh1, suggesting that Fhl1 does more than simply provide a platform for Ifh1. Tethering Ifh1 to the promoter leads to low-level transcription, at the correct initiation sites. Remarkably, activation by tethered LexA-Gal4(AD) is strongly reduced when TOR kinase is inhibited by rapamycin. Thus, TOR can act independently of Fhl1/Ifh1 at ribosomal protein promoters. We also show that, in our strain background, the response of ribosomal protein promoters to TOR inhibition is independent of the Ifh1-related protein Crf1, indicating that the role of this corepressor is strain specific. Fine-structure chromatin mapping of several ribosomal protein promoters revealed that histones are essentially absent from the Rap1 sites, while Fhl1 and Ifh1 are coincident with each other but distinct from Rap1.

Bacterial Proteins↗
PubMed2002

Protein kinase A operates a molecular switch that governs yeast pseudohyphal differentiation.

The yeast Saccharomyces cerevisiae undergoes a dimorphic filamentous transition in response to nutrient cues that is affected by both mitogen-activated protein kinase and cyclic AMP-protein kinase A signaling cascades. Here two transcriptional regulators, Flo8 and Sfl1, are shown to be the direct molecular targets of protein kinase A. Flo8 and Sfl1 antagonistically control expression of the cell adhesin Flo11 via a common promoter element. Phosphorylation by the protein kinase A catalytic subunit Tpk2 promotes Flo8 binding and activation of the Flo11 promoter and relieves repression by prohibiting dimerization and DNA binding by Sfl1. Our studies illustrate in molecular detail how protein kinase A combinatorially effects a key developmental switch. Similar mechanisms may operate in pathogenic fungi and more complex multicellular eukaryotic organisms.

Bacterial Proteins↗
PubMed2005

Rpm2p, a component of yeast mitochondrial RNase P, acts as a transcriptional activator in the nucleus.

Rpm2p, a protein subunit of yeast mitochondrial RNase P, has another function that is essential in cells lacking the wild-type mitochondrial genome. This function does not require the mitochondrial leader sequence and appears to affect transcription of nuclear genes. Rpm2p expressed as a fusion protein with green fluorescent protein localizes to the nucleus and activates transcription from promoters containing lexA-binding sites when fused to a heterologous DNA binding domain, lexA. The transcriptional activation region of Rpm2p contains two leucine zippers that are required for transcriptional activation and are conserved in the distantly related yeast Candida glabrata. The presence of a mitochondrial leader sequence does not prevent a portion of Rpm2p from locating to the nucleus, and several observations suggest that the nuclear location and transcriptional activation ability of Rpm2p are physiologically significant. The ability of RPM2 alleles to suppress tom40-3, a temperature-sensitive mutant of a component of the mitochondrial import apparatus, correlates with their ability to transactivate the reporter genes with lexA-binding sites. In cells lacking mitochondrial DNA, Rpm2p influences the levels of TOM40, TOM6, TOM20, TOM22, and TOM37 mRNAs, which encode components of the mitochondrial import apparatus, but not that of TOM70 mRNA. It also affects HSP60 and HSP10 mRNAs that encode essential mitochondrial chaperones. Rpm2p also increases the level of Tom40p, as well as Hsp60p, but not Atp2p, suggesting that some, but not all, nucleus-encoded mitochondrial components are affected.

Bacterial Proteins↗
PubMed2004

Identification of CED-3 substrates by a yeast-based screening method.

Identifying cellular substrates repertoire of individual proteases will facilitate our understanding of their physiological and pathological roles. In this article, we employed a yeast-based screening method to isolate CED-3 substrates. This method uses a transcription factor anchored to the plasma membrane by fusion to a library of cellular protein sequences. When a fusion protein is cleaved by CED-3, the transcription factor is released from the plasma membrane and enters the nucleus where it turns on the expression of reporter genes. We identified seven candidate clones by screening a genomic library using this method. Of these seven clones, two were cleaved by purified CED-3 in vitro. Therefore, the method described here may be generally used for genomewide screening to isolate potential substrates of specific proteases.

Bacterial Proteins↗
PubMed1995

Fos leucine zipper variants with increased association capacity.

The Fos wild-type leucine zipper is unable to support homodimerization. This finding is generally explained by the negative net charge of the Fos zipper leading to the electrostatic repulsion of two monomers. Using a LexA-dependent in vivo assay in Escherichia coli, we show here that additional antideterminants for Fos zipper association are the residues in position a within the Fos zipper interface. If the wild-type Fos zipper is fused to the DNA binding domain of the LexA repressor (LexA-DBD), no excess repression is observed as compared with the LexA-DBD alone, in agreement with the incapacity of the wild-type Fos zipper to promote homodimerization. If hydrophobic amino acids (Ile, Leu, Val, Phe, Met) are inserted into the five a positions of a LexA-Fos zipper fusion protein, substantial transcriptional repression is recovered showing that Fos zipper homodimerization is not only limited by the repulsion of negatively charged residues but also by the nonhydrophobic nature of the a positions. The most efficient variants (harboring Ile or Leu in the five a positions) show an about 80-fold increase in transcriptional repression as compared with the wild-type Fos zipper fusion protein. In the case of multiple identical substitutions, the overall improvement is correlated with the hydrophobicity of the inserted side chains, i.e. Ile Leu > Val > Phe > Met. However at least for Val, Phe, and Met the impact of a given residue type on the association efficiency depends strongly on the heptad, i.e. on the local environment of the a residue. This is particularly striking for the second heptad of the Fos zipper, where Val is less well tolerated than Phe and Met. Most likely the a1 residue modulates the interhelical repulsion between two glutamic acid side chains in positions g1 and e2. Most of the hydrophobic Fos zipper variants are also improved in heteroassociation with a Jun leucine zipper, such that roughly half of the additional free energy of homodimerization is imported into the heterodimer. A few candidates (including the Fos wild-type zipper) deviate from this correlation, showing considerable excess heteroassociation.

3T3 Cells↗
PubMedDate not supplied

A brief consideration of the SOS inducing signal.

SOS functions are induced in E. coli by treatments that damage cellular DNA or interrupt its synthesis. The biochemical basis of induction is activation of the specific proteolytic activity of recA protein, which then inactivates the lexA repressor. We discuss the development of the inducing signal in the cell.

Bacterial Proteins↗
PubMed2006

Functional domains of the Bacillus subtilis transcription factor AraR and identification of amino acids important for nucleoprotein complex assembly and effector binding.

The Bacillus subtilis AraR transcription factor represses at least 13 genes required for the extracellular degradation of arabinose-containing polysaccharides, transport of arabinose, arabinose oligomers, xylose, and galactose, intracellular degradation of arabinose oligomers, and further catabolism of this sugar. AraR exhibits a chimeric organization comprising a small N-terminal DNA-binding domain that contains a winged helix-turn-helix motif similar to that seen with the GntR family and a larger C-terminal domain homologous to that of the LacI/GalR family. Here, a model for AraR was derived based on the known crystal structures of the FadR and PurR regulators from Escherichia coli. We have used random mutagenesis, deletion, and construction of chimeric LexA-AraR fusion proteins to map the functional domains of AraR required for DNA binding, dimerization, and effector binding. Moreover, predictions for the functional role of specific residues were tested by site-directed mutagenesis. In vivo analysis identified particular amino acids required for dimer assembly, formation of the nucleoprotein complex, and composition of the sugar-binding cleft. This work presents a structural framework for the function of AraR and provides insight into the mechanistic mode of action of this modular repressor.

Amino Acid Sequence↗
PubMed1995

Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex.

The CCR4 protein from Saccharomyces cerevisiae is a component of a multisubunit complex that is required for the regulation of a number of genes in yeast cells. We report here the identification of a mouse protein (mCAF1 [mouse CCR4-associated factor 1]) which is capable of interacting with and binding to the yeast CCR4 protein. The mCAF1 protein was shown to have significant similarity to proteins from humans, Caenorhabditis elegans, Arabidopsis thaliana, and S. cerevisiae. The yeast gene (yCAF1) had been previously cloned as the POP2 gene, which is required for expression of several genes. Both yCAF1 (POP2) and the C. elegans homolog of CAF1 were shown to genetically interact with CCR4 in vivo, and yCAF1 (POP2) physically associated with CCR4. Disruption of the CAF1 (POP2) gene in yeast cells gave phenotypes and defects in transcription similar to those observed with disruptions of CCR4, including the ability to suppress spt10-enhanced ADH2 expression. In addition, yCAF1 (POP2) when fused to LexA was capable of activating transcription. mCAF1 could also activate transcription when fused to LexA and could functionally substitute for yCAF1 in allowing ADH2 expression in an spt10 mutant background. These data imply that CAF1 is a component of the CCR4 protein complex and that this complex has retained evolutionarily conserved functions important to eukaryotic transcription.

Amino Acid Sequence↗
PubMed1992

Formation of the LuxR protein in the Vibrio fischeri lux system is controlled by HtpR through the GroESL proteins.

The transcription of the luminescence (lux) system of Vibrio fischeri is regulated by the LuxR protein and an autoinducer. We previously showed that apart from these regulatory elements, the transcription of the lux system is negatively controlled by the LexA protein and positively controlled by the HtpR protein (sigma 32). This study was conducted in order to elucidate the mode of action of the HtpR protein. Using luxR-lacZ fused genes, we showed that the HtpR protein is essential for the maximum expression of beta-galactosidase activity in Escherichia coli lac mutant cells. Using this construct, we also demonstrated that luxR is preferentially expressed toward the end of the logarithmic phase of growth. Starvation and addition of ethanol significantly advanced the appearance of beta-galactosidase activity in htpR+ cells. The luminescence system of E. coli htpR+ cells harboring the pChv1 plasmid with a deletion in the luxI gene is induced in the presence of low and constant concentrations (150 pg/ml) of the inducer only at a late stage of the logarithmic phase of growth. When the cellular LuxR content is reduced, following 23 generations of exponential growth in Luria broth, a mid-log-phase culture does not respond to the inducer (150 pg/ml). On the basis of the above observations we suggest that the HtpR protein controls the formation of V. fischeri LuxR protein. Preliminary findings indicate that the HtpR protein acts through the chaperonins GroESL. E. coli htpR/pChv1 cells retained their full level of in vivo and in vitro luciferase activities in the presence of multiple copies of groESL genes. The possibility that GroESL proteins stabilize the native form of LuxR protein is discussed.

Bacterial Proteins↗
PubMed1982

Cleavage of lambda repressor and synthesis of RecA protein induced by transferred UV-damaged F sex factor.

Transfer of a UV-damaged F sex factor to a recipient lambda lysogen induces prophage lambda development. Under these conditions RecA protein synthesis was induced and lambda repressor cleaved, as observed upon direct induction, that is, when the recipient lambda lysogen was directly exposed to UV-light. The efficiency of induction of RecA protein synthesis in recipient bacteria which had received an irradiated F-lac factor was about 80% of that measured upon direct induction. We observed the simultaneous disappearance of lambda repressor and a slight production of cleavage fragments; quantitation by densitometric scanning of the autoradiogram after correction for the efficiency of transfer indicated that 55% of lambda repressor was cleaved. Transfer of UV-damaged Hfr DNA failed to induce RecA protein synthesis. A lambda phage vector carrying oriF, the cloned origin of F plasmid replication, after exposure to UV-light and infection of a recipient lysogen, induced RecA protein synthesis and a moderate but significant cleavage of lambda repressor. Indirect induction by UV-damaged F sex factor or phage lambda oriF resulted in biochemical cellular reactions similar to those observed upon direct induction. LexA repressor that negatively controls RecA protein synthesis appeared more susceptible to cleavage than did lambda repressor.

Bacterial Proteins↗
PubMed1998

The expression of recombinant genes from bacteriophage lambda strong promoters triggers the SOS response in Escherichia coli.

The production of several non-related heterologous proteins in recombinant Escherichia coli cells promotes a significant transcription of recA and sfiA SOS DNA repair genes. The activation of the SOS system occurs when the expression of plasmid-encoded genes is directed by the strong lambda lytic promoters, but not by IPTG-controlled promoters either at 37 or at 42 degrees C, and it is linked to an extensive degradation of the proteins after their synthesis. The triggering signal for the SOS response could be an important arrest of cell DNA replication observed within the first hour after the induction of recombinant gene expression. The stimulation of this DNA repair system can partially account for the toxicity exhibited by recombinant proteins on actively producing E. coli cells.

Bacterial Proteins↗
PubMed2006

Insights into the LexA regulon of Thermotogales.

The lexA genes of Thermotoga maritima and Petrotoga miotherma, both members of the Order Thermotogales, have been cloned and their transcriptional organization, as well as the functional characteristics of their encoded products, analyzed. In both bacterial species, the lexA gene was found to be co-transcribed together with another four (T. maritima) or three (P. miotherma) upstream open-reading frames. The P. miotherma LexA was able to bind promoters of both the cognate lexA encoding operon and the uvrA gene but not to that of the recA. Conversely, LexA protein and crude cell extracts from T. maritima were unable to bind promoters governing the expression of either its lexA or recA genes. In agreement with these observations, no functional copy of the P. miotherma LexA box, corresponding to the GANTN(6)GANNAC motif, seems to be present in the T. maritima genome. Giving support to the proposal that the evolutionary branching order of the Order Thermotogales is very close to that of Gram-positive bacteria, the P. miotherma LexA protein was still able to recognize the previously described LexA-binding sequence for Gram-positive bacteria.

Amino Acid Sequence↗
PubMed1987

The DNA-binding domains of the jun oncoprotein and the yeast GCN4 transcriptional activator protein are functionally homologous.

The jun oncoprotein, which causes sarcomas in chickens, and the DNA-binding domain of yeast GCN4, which coordinately regulates the expression of amino acid biosynthetic genes, show significant homology. In yeast cells deleted for the GCN4 gene, GCN4 function can be conferred by a hybrid protein in which the GCN4 DNA-binding domain is replaced by the homologous region of jun. Moreover, in strains containing various mutations of the GCN4 binding site in the HIS3 promoter, HIS3 expression is affected similarly by the hybrid protein and by GCN4. These results indicate that the jun oncoprotein binds the same DNA sequences as GCN4, and strongly suggest that jun is derived from a normal cellular transcription factor (possibly AP-1, which recognizes similar sequences). This provides direct evidence for the idea that alterations in the machinery for proper gene expression can lead to the oncogenic state.

Avian Sarcoma Viruses↗