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PubMed1992

Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers.

Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors.

Alcohol Dehydrogenase↗

PubMed1998

Expression and properties of wild-type and mutant forms of the Drosophila sex comb on midleg (SCM) repressor protein.

The Sex comb on midleg (Scm) gene encodes a transcriptional repressor of the Polycomb group (PcG). Here we show that SCM protein is nuclear and that its expression is widespread during fly development. SCM protein contains a C-terminal domain, termed the SPM domain, which mediates protein-protein interactions. The biochemical function of another domain consisting of two 100-amino-acid-long repeats, termed "mbt" repeats, is unknown. We have determined the molecular lesions of nine Scm mutant alleles, which identify functional requirements for specific domains. The Scm alleles were tested for genetic interactions with mutations in other PcG genes. Intriguingly, three hypomorphic Scm mutations, which map within an mbt repeat, interact with PcG mutations more strongly than do Scm null alleles. The strongest interactions produce partial synthetic lethality that affects doubly heterozygous females more severely than males. We show that mbt repeat alleles produce stable SCM proteins that associate with normal sites in polytene chromosomes. We also analyzed progeny from Scm mutant germline clones to compare the effects of an mbt repeat mutation during embryonic vs. pupal development. We suggest that the mbt repeat alleles produce altered SCM proteins that incorporate into and impair function of PcG protein complexes.

Alleles↗

PubMed1998

Molecular cloning of a novel transcriptional repressor protein of the rat type 1 vasoactive intestinal peptide receptor gene.

This study demonstrates that the transcriptional repressor sequence of the rat vasoactive intestinal peptide receptor (VIPR) gene constitutes a 42-base pair core element that is the binding site for a nuclear protein. We showed that this element was able to confer transcriptional repression to a heterologous promoter and that deletion or point mutations within this element resulted in loss of transcriptional repression. Southwestern blot analysis indicated that the VIPR repressor element interacts specifically with a nuclear protein of about 72 kDa. By screening a rat lung expression library coupled with rapid amplification of cDNA ends polymerase chain reactions, we isolated a cDNA clone (designated as VIPR-RP) that contains an open reading frame of 656 amino acids. VIPR-RP is 78% identical to a previously characterized protein, differentiation-specific element-binding protein, which is a member of a family of proteins including components of the DNA replication factor C complex. However, VIPR-RP cDNA encodes for a much smaller protein than differentiation-specific element-binding protein because of a frameshift. VIPR-RP mRNA is expressed in multiple tissues, including lung, liver, brain, heart, kidney, spleen, and testis. VIPR-RP protein specifically interacts with the VIPR repressor element as demonstrated by gel shift assays. Transfection of VIP-RP expression vector into Cos cells resulted in transcriptional repression of a reporter construct containing multiple copies of the VIPR repressor element. These results indicate that VIPR-RP is a novel transcriptional repressor protein that regulates VIPR expression.

Amino Acid Sequence↗

PubMed1999

The SNF1 kinase complex from Saccharomyces cerevisiae phosphorylates the transcriptional repressor protein Mig1p in vitro at four sites within or near regulatory domain 1.

Mig1p is a zinc finger protein required for repression of glucose-regulated genes in budding yeast. On removal of medium glucose, gene repression is relieved via a mechanism that requires the SNF1 protein kinase complex. We show that Mig1p expressed as a glutathione-S-transferase fusion in bacteria is readily phosphorylated by the SNF1 kinase in vitro. Four phosphorylation sites were identified, i.e. Ser-222, Ser-278, Ser-311 and Ser-381. The latter three are exact matches to the recognition motif we previously defined for SNF1 and lie within regions shown to be required for SNF1-dependent derepression and nuclear-to-cytoplasmic translocation.

Aspartic Acid↗

PubMed1996

The binding site of the IclR repressor protein overlaps the promoter of aceBAK.

In Escherichia coli, repression of the aceBAK operon is mediated by the IclR protein. We used an in vitro oligonucleotide selection technique to determine the consensus recognition sequence for MR. Mutational analysis confirmed the contribution of this sequence to repression in vivo and identified the -35 element of the promoter.

Bacterial Proteins↗

PubMed2001

Mass spectrometric study of the Escherichia coli repressor proteins, Ic1R and Gc1R, and their complexes with DNA.

In Escherichia coli, the IclR protein regulates both the aceBAK operon and its own synthesis. Database homology searches have identified many IclR-like proteins, now known as the IclR family, which can be identified by a conserved C-terminal region. We have cloned and purified one of these proteins, which we have named GclR (glyoxylate carboligase repressor). Although purification is straightforward, both the IclR and GclR proteins are difficult to manipulate, requiring high salt (up to 0.6 M KCl) for solubility. With the advent of nanospray ionization, we could transfer the proteins into much higher concentrations of volatile buffer than had been practical with ordinary electrospray. In 0.5 M ammonium bicarbonate buffer, both proteins were stable as tetramers, with a small amount of dimer. In a separate experiment, we found that IclR protein selected from a random pool a sequence which matched exactly that of the presumed binding region of the GclR protein, although IclR does not regulate the gcl gene. We designed a 29 bp synthetic DNA to which IclR and GclR bind, and with which we were able to form noncovalent DNA-protein complexes for further mass spectrometry analysis. These complexes were far more stable than the proteins alone, and we have evidence of a stoichiometry which has not been described previously with (protein monomer : dsDNA) = (4 : 1).

Bacterial Proteins↗

PubMed1999

A novel BTB/POZ transcriptional repressor protein interacts with the Fanconi anemia group C protein and PLZF.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome. The phenotype includes developmental defects, bone marrow failure, and cell cycle abnormalities. At least eight complementation groups (A-H) exist, and although three of the corresponding complementation group genes have been cloned, they lack recognizable motifs, and their functions are unknown. We have isolated a binding partner for the Fanconi anemia group C protein (FANCC) by yeast two-hybrid screening. We show that the novel gene, FAZF, encodes a 486 amino acid protein containing a conserved amino terminal BTB/POZ protein interaction domain and three C-terminal Krüppel-like zinc fingers. FAZF is homologous to the promyelocytic leukemia zinc finger (PLZF) protein, which has been shown to act as a transcriptional repressor by recruitment of nuclear corepressors (N-CoR, Sin3, and HDAC1 complex). Consistent with a role in FA, BTB/POZ-containing proteins have been implicated in oncogenesis, limb morphogenesis, hematopoiesis, and proliferation. We show that FAZF is a transcriptional repressor that is able to bind to the same DNA target sequences as PLZF. Our data suggest that the FAZF/FANCC interaction maps to a region of FANCC deleted in FA patients with a severe disease phenotype. We also show that FAZF and wild-type FANCC can colocalize in nuclear foci, whereas a patient-derived mutant FANCC that is compromised for nuclear localization cannot. These results suggest that the function of FANCC may be linked to a transcriptional repression pathway involved in chromatin remodeling.

Amino Acid Sequence↗

PubMed1997

Evolutionary conservation and predicted structure of the Drosophila extra sex combs repressor protein.

The Drosophila extra sex combs (esc) protein, a member of the Polycomb group (PcG), is a transcriptional repressor of homeotic genes. Genetic studies have shown that esc protein is required in early embryos at about the time that other PcG proteins become engaged in homeotic gene repression. The esc protein consists primarily of multiple copies of the WD repeat, a motif that has been implicated in protein-protein interaction. To further investigate the domain organization of esc protein, we have isolated and characterized esc homologs from divergent insect species. We report that esc protein is highly conserved in housefly (72% identical to Drosophila esc), butterfly (55% identical), and grasshopper (56% identical). We show that the butterfly homolog provides esc function in Drosophila, indicating that the sequence similarities reflect functional conservation. Homology modeling using the crystal structure of another WD repeat protein, the G-protein beta-subunit, predicts that esc protein adopts a beta-propeller structure. The sequence comparisons and modeling suggest that there are seven WD repeats in esc protein which together form a seven-bladed beta-propeller. We locate the conserved regions in esc protein with respect to this predicted structure. Site-directed mutagenesis of specific loops, predicted to extend from the propeller surface, identifies conserved parts of esc protein required for function in vivo. We suggest that these regions might mediate physical interaction with esc partner proteins.

Amino Acid Sequence↗

PubMed1972

Detection and isolation of the repressor protein for the tryptophan operon of Escherichia coli.

DNA from a transducing bacteriophage carrying a fusion of the tryptophan and lactose operons of E. coli (lambdadtrp-lac) has been used to direct cell-free synthesis of beta-galactosidase (EC 3.2.1.23). Whereas normal lac operon (lambdadlac) DNA requires adenosine-3':5'-cyclic monophosphate (cAMP) for beta-galactosidase synthesis, trp-lac DNA is unaffected by cAMP. This difference in cAMP dependence verifies the presence of a cAMP-requiring promoter in the lac operon that has been removed from the trp-lac DNA. Synthesis with trp-lac DNA is controlled by the protein product of the tryptophan repressor gene (trpR). Synthesis in extracts of trpR(-) (repressor-negative) cells is progressively reduced by increased additions of extract from trpR(+) cells. No trpR(-) product repression is seen when beta-galactosidase synthesis is programmed by normal lac DNA. This highly sensitive and specific assay has facilitated quantitation and partial purification of the trp repressor.

Bacterial Proteins↗

PubMedDate not supplied

A nuclear magnetic resonance study of the DNA-binding affinity of Cro repressor protein stabilized by a disulfide bond.

The structure, dynamics, and DNA-binding characteristics of wild-type and cross-linked Cro repressors are compared by using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The Cro repressor is a small dimeric DNA-binding protein from bacteriophage lambda. Replacement of valine-55 by cysteine in the dimer interaction region of each monomer subunit results in the spontaneous formation of a disulfide cross-link between the subunits. Two-dimensional nuclear Overhauser effect spectroscopy and CD data show the variant has nearly the same conformation as the wild-type protein. However, by monitoring the CD band at 222 nm, the cross-linked protein is shown to have a heat-denaturation midpoint temperature of 67 degrees C, whereas the wild-type protein has a melting temperature of about 47 degrees C. Using 1H-NMR to follow the denaturation by heat, the same melting temperature is observed for wild-type Cro (47 degrees C), but a much lower melting temperature is seen for V55C Cro (58 degrees C). This suggests that between 58 and 67 degrees C the cross-linked protein exists in a molten globule state with the alpha-helices mainly intact, but without the interaction of chemical groups that cause spectral dispersion. Binding parameters for interaction of the proteins with DNA were obtained by observing the NMR spectrum for the imino protons of a 10 base-pair half-operator DNA and titrating in protein. The cross-linked protein binds DNA (Kd = 160 microM) about eight times more weakly than the wild-type protein (Kd = 19 microM). Adjustments in protein structure, necessary to form a tight protein-DNA complex, appear to be hindered by a loss in protein flexibility caused by the intersubunit cross-link.

Bacteriophage lambda↗

PubMed1996

DNA-binding properties of the BetI repressor protein of Escherichia coli: the inducer choline stimulates BetI-DNA complex formation.

The betT and betIBA genes govern glycine betaine synthesis from choline in Escherichia coli. In an accompanying paper we report that the betT and betI promoters are divergently organized and partially overlapping and that both are negatively regulated by BetI in response to choline. (T. Lamark, T.P. Rokenes, J. McDougall, and A.R. Strom, J. Bacteriol. 178:1655-1662, 1996). In this paper, we report that the in vivo synthesis rate of the BetI protein constituted only 10% of that of BetA and BetB dehydrogenase proteins, indicating the existence of a posttranscriptional control of the betIBA operon. A genetically modified BetI protein called BetI*, which carries 7 extra N-terminal amino acids, was purified as a glutathione S-transferase fusion protein. Gel mobility shift assays showed that BetI* formed a complex with a 41-bp DNA fragment containing the -10 and -35 regions of both promoters. Only one stable complex was detected with the 41-bp fragment and all larger promoter-containing fragments tested. In DNase I footprinting, BetI* protected a region of 21 nucleotides covering both the -35 boxes. Choline stimulated complex formation but did not change the binding site of BetI*. We conclude that in vivo BetI is bound to its operator in both repressed and induced cells and that BetI represents a new type of repressor.

Bacterial Proteins↗

PubMed2000

A direct photo-activated affinity modification of tetracycline transcription repressor protein TetR(D) with tetracycline(1).

Results of a first successful application of a direct photo-induced affinity modification of Tet repressor (TetR(D)) protein with tetracycline within a complex of known three-dimensional structure are described. The conditions of the modification have provided suitable yields of the modified complex and allowed characterization of the modified segments of the protein. The potential of tetracycline as a fine modifying reagent was established. In the complex of TetR(D) protein with tetracycline, the antibiotic modifies at least two segments, Ile59-Glu73 and Ala173-Glu183, which form a binding tunnel for the drug according to the X-ray analysis. These data open possibilities for the use of different tetracycline targets for structural studies in solution.

Amino Acid Sequence↗

PubMed2005

Role of USF1 and USF2 as potential repressor proteins for human intestinal monocarboxylate transporter 1 promoter.

Butyrate, a short-chain fatty acid, is the major energy fuel for the colonocytes. We have previously reported that monocarboxylate transporter isoform 1 (MCT1) mediates uptake of butyrate by human colonic Caco-2 cells. To better understand the mechanisms of MCT1 expression and regulation in the human intestine, we examined the activity and regulation of MCT1 promoter in Caco-2 cells. The transcription initiation site in the MCT1 promoter was identified as a guanine nucleotide 281 bp upstream from the translation initiation site and is surrounded by a guanine-cytosine-rich area. The promoter was found to be highly active when transfected into Caco-2 cells, and its activity decreased with deletions at its 5'-end. Gel mobility shift experiments showed binding of the transcription factors upstream stimulatory factor (USF)1 and 2 to the site -114 to -119 of the MCT1 promoter. With the use of site-directed mutagenesis and promoter activity in Caco-2 cells, the USF proteins appeared to have a repressor role on the MCT1 promoter, which was further confirmed by cotransfecting expression vectors encoding USF1 and 2 in Caco-2 cells and determining endogenous MCT1 expression in USF2 overexpressed cells. The two potential SP1 binding sites found in the same region of the promoter were found not to be involved in its regulation.

Base Sequence↗

PubMed1999

RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1.

The products of the Polycomb group (PcG) of genes are necessary for the maintenance of transcriptional repression of a number of important developmental genes, including the homeotic genes. A two-hybrid screen was used to search for putative new members of the PcG of genes in mammals. We have identified a new Zn finger protein, RYBP, which interacts directly with both Ring1 proteins (Ring1A and Ring1B) and with M33, two mutually interacting sets of proteins of the mammalian Polycomb complex. Ring1 binds RYBP and M33 through the same C-terminal domain, whereas the RYBP-M33 interaction takes place through an M33 domain not involved in Ring1 binding. RYBP also interacts directly with YY1, a transcription factor partially related to the product of the Drosophila pleiohomeotic gene. In addition, we show here that RYBP acts as a transcriptional repressor in transiently transfected cells. Finally, RYBP shows a dynamic expression pattern during embryogenesis which initially overlaps partially that of Ring1A in the central nervous system, and later becomes ubiquitous. Taken together, these data suggest that RYBP may play a relevant role in PcG function in mammals.

Amino Acid Sequence↗

PubMed1987

Thermodynamic analysis of inducer binding to the lactose repressor protein: contributions of galactosyl hydroxyl groups and beta substituents.

Kinetic and equilibrium studies of the binding of modified beta-D-galactoside sugars to the lac repressor were carried out to generate thermodynamic data for protein-inducer interactions. The energetic contributions of the galactosyl hydroxyl groups to binding were assessed by using a series of methyl deoxyfluoro-beta-D-galactosides. The C-3 and C-6 hydroxyls contributed less than or equal to -2.3 and -1.7 +/- 0.3 kcal/mol to the binding free energy change, respectively, whereas the C-4 hydroxyl provided only a nominal contribution (-0.1 +/- 0.2 kcal/mol). Favorable contributions to the total binding free energy change were observed for replacement of O-methyl by S-methyl at the beta-anomeric position and for S-methyl by S-isopropyl. Negative delta H degrees values characteristic of protein-sugar complexes [Quiocho, F. A. (1986) Annu. Rev. Biochem. 55, 287-315] were observed for a series of beta-D-galactosides differing at the beta-glycosidic position. A decrease in delta H degrees of approximately 6 kcal/mol upon replacement of the O-methyl substituent by S-methyl indicates a substantial increase in van der Waals' interactions and/or hydrogen bonding in this region of the ligand binding site. The more favorable free energy change for the binding of the S-isopropyl vs S-methyl compound is due mainly to more positive entropic contributions, consistent with an increase in apolar interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Galactose↗

PubMed1992

Regions of bacteriophage T4 and RB69 RegA translational repressor proteins that determine RNA-binding specificity.

RegA protein of T4 and related bacteriophages is a highly conserved RNA-binding protein that represses the translation of many phage mRNAs that encode enzymes involved in DNA metabolism. RB69, a T4-related bacteriophage, has a unique regA gene, which we have cloned, sequenced, and expressed. The predicted amino acid sequence of RB69 RegA is 78% identical to that of T4 RegA. Plasmid-encoded RB69 RegA expressed in vivo represses the translation of T4 early mRNAs, including those of rIIA, rIIB, 44, 45, rpbA, and regA. Nucleotide sequences were determined for several T4 and RB69 regA mutations, and their corresponding repressor properties were characterized. All of the 10 missense mutations affect residues conserved between RB69 and T4 RegA. Two regions of RegA are especially sensitive to mutation: one between Val-15 and Ala-25 and another between Arg-70 and Ser-73. Sequence alignments and mutational data suggest that the region from Val-15 to Ala-25 is similar to helix-turn-helix domains of DNA-binding proteins and confers RNA-binding specificity upon RegA. The RegA691 protein (Ile-24----Thr) has an in vivo phenotype that appears to distinguish site-specific and cooperative binding modes of hierarchical RegA-mediated translational repression.

Amino Acid Sequence↗

PubMed1995

SARs are cis DNA elements of chromosome dynamics: synthesis of a SAR repressor protein.

SARs are candidate DNA elements for defining the bases of chromatin loops and possibly for serving as cis elements of chromosome dynamics. SARs contain numerous A tracts, whose altered DNA structure is recognized by cooperatively interacting proteins such as topoisomerase II. We constructed multi-AT hook (MATH) proteins and demonstrate that they specifically bind the clustered A tracts of SARs in chromatin and chromosomes. They are also potent inhibitors of chromosome assembly in mitotic Xenopus extracts, demonstrating the importance of SARs in this process. Titration of SARs with MATH20 (20 hooks) blocks shape determination of chromatids but not chromatin condensation per se. SARs are also required for shape maintenance of chromosomes. If MATH20 is added after formation of chromatids, they collapse and are reshaped by an active, mitotic process into spherical chromatid balls.

Adenine↗

PubMed1980

Hybrid tetramers of native and core lactose repressor protein. Assessment of operator and nonspecific DNA binding parameters and their relationship.

Hybrid tetramers of lac repressor and its trypsin-resistant core protein were produced by mild proteolytic digestion and isolated by chromatography on phosphocellulose. These tetramers were used in binding studies to probe the relationship of the NH2 terminus and core domains in operator DNA binding and to explore subunit participation in both operator and nonspecific DNA binding. The purity of each tetramer was demonstrated by several lines of evidence, including physical characterization and measurement of the binding activities of the hybrid tetramer preparations. Each tetramer displayed measurable operator binding activity, and the dissociation constants of the tetramers for lambdaplac DNA and a 29 base operator fragment increase with the loss of each NH2 terminus. These studies strongly suggest that each NH2 terminus of the repressor interacts with the operator DNA, although the four NH2 termini do not appear to make equal contacts. The contributions of the NH2 terminus to operator binding do no appear to be identical with those for nonspecific DNA binding. In addition, the binding of the NH2 termini to nonspecific DNA can be described by postulating four identical, noninteracting sites. Although the rate of dissociation of the repressor-operator is increased by a factor of 2 upon removal of each NH2 terminus, calculations using the measured dissociation rate and equilibrium dissociation constant indicate that the rate of association of repressor to operator is decreased approximately 5-fold. These data suggest that the contacts made by the NH2 termini may greatly facilitate association with DNA. Competition studies of nonspecific DNA with operator DNA binding confirm the existence of two operator DNA binding sites on each tetramer and suggest that the contacts of a single NH2 terminus are not identical for the two operator binding sites.

Binding, Competitive↗