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Results for “repressor proteins”

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PubMed2001

Multiple levels of control regulate the yeast cAMP-response element-binding protein repressor Sko1p in response to stress.

The Sko1p transcriptional repressor regulates a subset of osmoinducible stress defense genes in Saccharomyces cerevisiae by binding to cAMP-responsive elements. We have reported previously that in response to stress Sko1p is phosphorylated by the stress-activated Hog1p mitogen-activated protein kinase, which disrupts its interaction with the Ssn6p x Tup1p corepressor. Here we report that other mechanisms are essential for the regulation of the Sko1p repressor activity upon stress. The nuclear localization of Sko1p depends on the stress-inhibited protein kinase A (PKA). Sko1p is localized in the nucleus of unstressed cells, and it redistributes to the cytosol upon severe salt stress (1 m NaCl). Yeast mutants with low PKA activity localize Sko1p to the cytoplasm in the absence of stress and exhibit deregulated expression of cAMP-responsive element-regulated genes. The central part (315) of Sko1p, containing the PKA phosphorylation sites and the basic domain-leucine zipper domain, is essential for its nuclear localization. Salt-induced export of Sko1p from the nucleus is independent of Hog1p and of the Bcy1p regulatory subunit of PKA. Furthermore, phosphorylation by PKA slightly enhanced DNA binding affinity of Sko1p in vitro, whereas Sko1p dimerization in vivo is not regulated by stress. Sko1p repressor activity is associated to its binding to the Ssn6p x Tup1p complex. Interestingly, the Sko1p NH(2) terminus (1), containing the Hog1p phosphorylation sites, associates in vivo with Tup1p in the absence of Ssn6p, suggesting that Sko1p represses gene transcription by interacting directly with the Tup1p subunit of the Ssn6p x Tup1p complex.

Basic-Leucine Zipper Transcription Factors↗

PubMed1997

Regulation of the dnaK operon of Streptomyces coelicolor A3(2) is governed by HspR, an autoregulatory repressor protein.

The dnaK operon of Streptomyces coelicolor contains four genes (5'-dnaK-grpE-dnaJ-hspR). The fourth gene encodes a novel heat shock protein, HspR, which appears so far to be unique to the high-G+C actinomycete group of bacteria. HspR binds with high specificity to three inverted repeat sequences in the promoter region of the S. coelicolor dnaK operon, strongly suggesting a direct role for HspR in heat shock gene regulation. Here we present genetic and biochemical evidence that HspR is the repressor of the dnaK operon. Disruption of hspR leads to high-level constitutive transcription of the dnaK operon. Parallel transcriptional analyses of groESL1 and groEL2 expression demonstrated that heat shock regulation of the groE genes was essentially unaffected in an hspR null mutant, although the basal (uninduced) level of groEL2 transcription was slightly elevated compared with the wild type. The results of HspR titration experiments, where the dnaK operon promoter region was cloned at ca. 50 copies per chromosome, were consistent with the prediction that HspR functions as a negative autoregulator. His-tagged HspR, overproduced and purified from Escherichia coli, was shown to repress transcription from the dnaK operon promoter in vitro, providing additional evidence for the proposal that HspR directly regulates transcription of the dnaK operon. These studies indicate that there are at least two transcriptional mechanisms for controlling heat shock genes in S. coelicolor--one controlling the dnaK operon and another controlling the groE genes.

Bacterial Proteins↗

PubMed1976

Kinetic studies of inducer binding to lactose repressor protein.

The kinetics of binding of the inducer, isopropyl-beta, D-thiogalactoside to lactose repressor from Escherichia coli was studied by stopped flow rapid mixing techniques. Three different spectral probes for measuring changes in the conformation of the repressor were used: ultraviolet absorbance, fluorescence, and a reporter group, 2-mercuri-4-nitrophenol, in the visible region. Repressor can be reacted with this mercurial to modify two of the three free sulfhydryl groups per monomer without loss of inducer or operator binding activities. The observed first order rate constant for the reaction of repressor with 2-chloromercuri-4-nitrophenol at pH 7.5 and 20 degrees was found to be on the order of 0.1 s-1, an unexpectedly slow rate for this type of reaction. Once bound to repressor, the nitrophenol serves as a chromophoric probe to monitor changes in the surrounding environment. The binding of inducer to repressor causes a change in the absorbance of the bound 2-mercuri-4-nitrophenol moiety and exhibits a second order rate of 3.2 x 10(4) M-1 s-1. Similar rates were obtained when binding of inducer is monitored by changes in either the ultraviolet absorbance of fluorescence of tryptophan residues. Since the same rate of spectral perturbation is observed for different regions of the primary structure of the protein, the conformational change produced in response to inducer binding appears to be translated rapidly throughout the protein molecule.

Bacterial Proteins↗

PubMed1998

QacR is a repressor protein that regulates expression of the Staphylococcus aureus multidrug efflux pump QacA.

The Staphylococcus aureus QacA protein is a multidrug transporter that confers resistance to a broad range of antimicrobial agents via proton motive force-dependent efflux of the compounds. Primer extension analysis was performed to map the transcription start points of the qacA and divergently transcribed qacR mRNAs. Each gene utilized a single promoter element, the locations of which were confirmed by site-directed mutagenesis. Fusions of the qacA and qacR promoters to a chloramphenicol acetyl transferase reporter gene were used to demonstrate that QacR is a trans-acting repressor of qacA transcription that does not autoregulate its own expression. An inverted repeat overlapping the qacA transcription start site was shown to be the operator sequence for control of qacA gene expression. Removal of one half of the operator prevented QacR-mediated repression of the qacA promoter. Purified QacR protein bound specifically to this operator sequence in DNase I-footprinting experiments. Importantly, addition of diverse QacA substrates was shown to induce qacA expression in vivo, as well as inhibit binding of QacR to operator DNA in vitro, by using gel-mobility shift assays. QacR therefore appears to interact directly with structurally dissimilar inducing compounds that are substrates of the QacA multidrug efflux pump.

Amino Acid Sequence↗

PubMed1990

Gene F of plasmid RSF1010 codes for a low-molecular-weight repressor protein that autoregulates expression of the repAC operon.

The repAC operon of plasmid RSF1010 consists of the genes for proteins E, F, RepA (DNA helicase), and RepC (origin-binding initiator protein) and is transcriptionally initiated by a promoter called P4. We have studied the expression of the repAC operon in vivo by using fusions to the lacZ reporter gene. The results show that the product of the second gene, F, autoregulates the operon by inhibiting transcription from P4. To verify its properties postulated from the in vivo studies and to initiate its biochemical characterization, we have purified the F protein from an overproducing E.coli strain constructed in vitro. Purification was based on a gel retardation assay for detection of P4-specific DNA binding. Subsequent DNase footprinting of the F binding sites showed clear protection around two partially symmetric P4 sequences of 16 bp, each of which matches the symmetric consensus sequence, GCGTGAGTACTCACGC, in at least 13 positions. The native repressor, as judged from gel filtration, velocity sedimentation and crosslinking studies, exists as a dimer in dilute solution; its monomeric subunit, as predicted from DNA sequence and N-terminal protein sequence data, consists of 68 amino acids and has a calculated M tau = 7,673.

Base Sequence↗

PubMed2002

Role of RegM, a homologue of the catabolite repressor protein CcpA, in the virulence of Streptococcus pneumoniae.

As part of a study of virulence gene regulation in Streptococcus pneumoniae, we have identified a gene encoding a homologue of the staphylococcal catabolite control protein CcpA in the pneumococcal genome sequence. The pneumococcal protein, designated RegM, has significant similarity to members of the LacI/GalR family of bacterial regulatory proteins. S. pneumoniae D39 derivatives with insertion-duplication or deletion mutations in regM were significantly attenuated in virulence with respect to the wild-type strain. In defined media containing either sucrose or lactose as sole carbon sources, the in vitro growth rates of D39 and the regM mutants were essentially the same. However, in the presence of galactose the regM mutants grew significantly faster than the wild-type strain, whereas growth rates were significantly lower in the presence of glucose or maltose. These data are consistent with the involvement of regM in the catabolism of carbohydrates in S. pneumoniae. RegM was a repressor of both alpha-glucosidase and beta-galactosidase activities in S. pneumoniae, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose. The observed attenuation in virulence was not attributable to poorer growth of the regM mutants in mouse blood ex vivo, but nevertheless, the mutants were rapidly cleared from the blood of infected mice in vivo. The regM mutation had no apparent impact on expression of several confirmed pneumococcal virulence proteins, but studies employing a lacZ transcriptional fusion construct indicated that mutation of regM resulted in a significant reduction in transcription of the capsular polysaccharide biosynthesis locus (cps). Thus, regM is the first gene outside of the cps locus to be implicated in regulation of capsular gene expression.

Amino Acid Sequence↗

PubMed1972

Altered sequences changing the operator-binding properties of the Lac repressor: colinearity of the repressor protein with the i-gene map.

A technique is described for mapping point mutations in the first 59 amino-acid residues of the lac repressor from Escherichia coli, using less than 0.1 mumol (4 mg) of the purified protein. This technique was used to localize five mutations affecting the ability of the i-gene product to repress in vivo. These alterations are located at four different sites in the amino-terminal region of the repressor molecule. Three of these are missense mutations and result in changes from serine to proline (residue 16), threonine to alanine (residue 19), and alanine to valine (residue 53). Each amino-acid substitution alone is sufficient to eliminate repression in vivo, presumably by altering the operator binding activity. The remaining two independently-isolated mutations are identical, and result in a change from a glutamine codon at position 26 to an amber (UAG) codon. Since suppression of this nonsense mutation with amber suppressors that insert leucine, tyrosine, serine, or glutamine restores repressor activity to the molecule, glutamine(26) cannot be crucial for the operator-binding function. A comparison of the position of each altered residue with the genetic map enabled us to estimate the physical distance between several deletion-group endpoints.

Amino Acid Sequence↗

PubMed2005

Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding.

The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task.

Bacterial Proteins↗

PubMed1997

Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle. Isolation of a cDNA clone for repressor protein Nished.

The expression of the cardiac myosin light chain 2 (MLC2) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (-360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to DeltaCSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor "runt," a known repressor of gap and pair-rule gene expression in Drosophila.

Amino Acid Sequence↗

PubMed1973

Isolation of the self-regulated repressor protein of the Hut operons of Salmonella typhimurium.

In Salmonella typhimurium the structural genes of the enzymes responsible for histidine utilization (hut) are clustered in two adjacent operons. A single repressor regulates both operons. The repressor itself is a member of one of the hut operons and, thus, regulates its own synthesis. We have assayed the hut repressor by its ability to bind radioactive DNA to nitrocellulose filters. The binding is specific for DNA bearing the hut operons, and the binding is abolished by the inducer, urocanate. As a member of one of the hut operons, the repressor is inducible, subject to catabolite repression, and affected by a promoter mutation.

Acrylates↗

PubMed2003

Truncated estrogen receptor product-1 stimulates estrogen receptor alpha transcriptional activity by titration of repressor proteins.

The truncated estrogen receptor product-1 (TERP-1, or TERP) is a pituitary-specific isoform of estrogen receptor alpha (ERalpha), and its expression is regulated by estrogen. TERP modulates the transcriptional activity of ERalpha but has no independent effect on transcription of estrogen-response element-containing promoters. At low concentrations, TERP stimulates ERalpha transcriptional activity in transient transfection assays. At TERP concentrations equal to or greater than full-length ERalpha, TERP forms dimers with ERalpha and reduces both ligand-dependent and -independent transcription. A dimerization mutant of TERP, TERP L509R, stimulated ERalpha transcription at all concentrations. We hypothesized that TERP stimulates ERalpha transcriptional activity by titrating suppressors of ERalpha activity. We found that repressor of estrogen receptor activity (REA), originally isolated from human breast cancer cells, is present in mouse pituitary gonadotrope cell lines. Levels of REA vary slightly throughout the rat reproductive cycle, but TERP mRNA and protein vary much more dramatically. In transfection experiments, REA suppressed ERalpha transcriptional activity, and TERP L509R was able to alleviate transcriptional suppression by REA. In glutathione S-transferase pull-down assays, TERP bound to REA more efficiently than did ERalpha at equivalent concentrations, suggesting that REA will preferentially bind to TERP. Our findings suggest that the stimulation of pituitary ERalpha activity by low concentrations of TERP can occur by titration of corepressors such as REA.

Animals↗

PubMed1973