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PubMedDate not supplied

Negative control at a distance mediates catabolite repression in yeast.

In prokaryotic organisms, the control of gene expression is mediated by regulatory proteins that activate or repress transcription. However, the molecular mechanisms of positive and negative control are different. In terms of negative control, repressor proteins bind to sites located within the promoter region and as a consequence sterically interfere with functional binding by RNA polymerase. Here, I examine the properties of a regulatory sequence that specifies catabolite (glucose) repression in the yeast Saccharomyces cerevisiae. Specifically, a DNA segment containing this regulatory site was fused upstream of the intact his3 promoter region and structural gene at several locations. Normally, his3 expression in these derivatives occurs at a basal level which can be induced by conditions of amino-acid starvation. However, in glucose medium, the catabolite regulatory sequence overrides the normal his3 promoter elements and reduces transcription both in normal and starvation conditions. The implication from these results is that in contrast to catabolite repression in Escherichia coli, which is mediated by catabolite-activating protein (CAP), catabolite repression in yeast occurs by a negative control mechanism involving a putative repressor protein. The observation that this regulatory site exerts its repressing effects even when located upstream of an intact promoter region suggests that repression in yeast is not mediated by steric interference between regulatory proteins and the transcriptional apparatus.

DNA, Recombinant↗

PubMed2003

GLIS3, a novel member of the GLIS subfamily of Krüppel-like zinc finger proteins with repressor and activation functions.

In this study, we describe the identification and characterization of a novel transcription factor GLI-similar 3 (GLIS3). GLIS3 is an 83.8 kDa nuclear protein containing five C2H2-type Krüppel-like zinc finger motifs that exhibit 93% identity with those of GLIS1, however, little homology exists outside their zinc finger domains. GLIS3 can function as a repressor and activator of transcription. Deletion mutant analysis determined that the N- and C-termini are required for optimal transcriptional activity. GLIS3 binds to the GLI-RE consensus sequence and is able to enhance GLI-RE-dependent transcription. GLIS3(DeltaC496), a dominant-negative mutant, inhibits transcriptional activation by GLIS3 and GLI1. Whole mount in situ hybridization on mouse embryos from stage E6.5 through E14.5 demonstrated that GLIS3 is expressed in specific regions in developing kidney and testis and in a highly dynamic pattern during neurulation. From E11.5 through E12.5 GLIS3 was strongly expressed in the interdigital regions, which are fated to undergo apoptosis. The temporal and spatial pattern of GLIS3 expression observed during embryonic development suggests that it may play a critical role in the regulation of a variety of cellular processes during development. Both the repressor and activation functions of GLIS3 may be involved in this control.

Amino Acid Sequence↗

PubMed1996

Purification and self-association equilibria of the lysis-lysogeny switch proteins of coliphage 186.

The CI repressor protein, responsible for maintenance of the lysogenic state, and the Apl protein, required for efficient prophage induction, are the two control proteins of the lysis-lysogeny transcriptional switch of coliphage 186. These proteins have been overexpressed, purified, and their self-association behavior examined by sedimentation equilibrium. Phage 186 CI dimers self-associate in solution through tetramers to octamers in a concerted process. The Apl protein of 186 is an unusual example of a helix-turn-helix protein which is monomeric in solution.

Base Sequence↗

PubMed1979

Degradation of the DNA-binding domain of wild-type and i-d lac repressors in Escherichia coli.

It has been shown that 28 transdominant mutant lac repressors which have lost operator DNA-binding ability in vivo and in vitro, but still bind inducer and are able to form tetramers (i-d repressors), could be divided into two groups by their capacity or incapacity to bind non-specifically to the phosphate groups of the DNA backbone. All but one of 15 analysed i-d repressors with amino acid substitutions to the C-terminal of residue 52 showed uneffected non-specific DNA binding. All 13 tested i-d repressors with amino acid substitutions to the N-terminal of residue 53 did not bind to double-stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene were endogenously degraded. The degradation in vivo only affects the amino-terminal 50-60 residues producing a mutant-specific pattern of stable repressor fragments. These fragments are tetrameric and capable of binding inducer in vivo and in vitro. The proteolytic attack presumably takes place during synthesis of the i-d repressors, since the resulting fragments are stable, both in vivo (as shown by a pulse-chase experiment) and in vitro. The proteolysis in vivo depends on the growth conditions of the bacteria and is higher in cells grown in minimal media than in rich media. Wild-type repressor is only susceptible to limited proteolysis in cells grown in minimal media but not in cells grown in rich media. The results suggest that the majority of the sequence alterations before residue 53 in missense mutant i-d lac repressor proteins affect the three-dimensional structure of the amino-terminal DNA-binding domain of the repressor protein, making it susceptible to proteolytic attack by one or several intracellular proteases.

Amino Acid Sequence↗

PubMedDate not supplied

Transposition protein of Tn3: identification and characterisation of an essential repressor-controlled gene product.

Fusions that bring lac gene expression under the control of transcriptional and translational signals within the Tn3 element have been used to identify and characterise a Tn3-encoded 'transposase' (Tnp) peptide of MW 100,000 essential for transposition. The gene specifying this product is regulated by the Tn3 repressor protein and is part of a bidirectional genetic unit that includes the repressor gene.

Bacterial Proteins↗

PubMed1989

Effects of carriage and expression of the Tn10 tetracycline-resistance operon on the fitness of Escherichia coli K12.

We have been examining the consequences of alternative modes of regulation of plasmid-borne, Tn10-encoded tetracycline resistance for the fitness of Escherichia coli. In a tetracycline-free environment, we measured the effects on fitness that were caused by (1) maximally induced expression of the resistance operon, (2) low-level constitutive expression of the resistance protein, (3) residual expression of the repressed resistance operon, (4) carriage of the resistance operon, (5) the remainder of the plasmid genome, and (6) hyperexpression of the repressor protein. We observed large reductions in fitness that were associated with induction and with constitutive expression of the tetracycline-resistance protein, but there was no discernible effect of hyperexpression of the repressor protein. We also observed a small reduction in fitness associated with the remainder of the plasmid genome. However, any reductions in fitness that were caused by residual expression and by carriage of the repressed operon were not more than 0.3%. We conclude that tight gene regulation has eliminated antagonistic pleiotropic effects of the resistance gene on fitness, so that possession of an inducible Tn10-encoded tetracycline-resistance operon imposes essentially no burden in the absence of antibiotic.

DNA Transposable Elements↗

PubMed1996

Activation mechanism of the multifunctional transcription factor repressor-activator protein 1 (Rap1p).

Transcriptional activation in eukaryotic organisms normally requires combinatorial interactions of multiple transcription factors. In most cases, the precise role played by each transcription factor is not known. The upstream activating sequence (UAS) elements of glycolytic enzyme genes in Saccharomyces cerevisiae are excellent model systems for the study of combinatorial interactions. The yeast protein known as Rap1p acts as both a transcriptional repressor and an activator, depending on sequence context. Rap1p-binding sites are found adjacent to Gcr1p-binding sites in the UAS elements of glycolytic enzyme genes. These UAS elements constitute some of the strongest activating sequences known in S. cerevisiae. In this study, we have investigated the relationship between Rap1p- and Gcr1p-binding sites and the proteins that bind them. In vivo DNA-binding studies with rap1ts mutant strains demonstrated that the inability of Rap1p to bind at its site resulted in the inability of Gcr1p to bind at adjacent binding sites. Synthetic oligonucleotides, modeled on the UAS element of PYK1, in which the relative positions of the Rap1p- and Gcr1p-binding sites were varied prepared and tested for their ability to function as UAS elements. The ability of the oligonucleotides to function as UAS elements was dependent not only on the presence of both binding sites but also on the relative distance between the binding sites. In vivo DNA-binding studies showed that the ability of Rap1p bind its site was independent of Gcr1p but that the ability of Gcr1p to bind its site was dependent on the presence of an appropriately spaced and bound Rap1p-binding site. In vitro binding studies showed Rap1p-enhanced binding of Gcr1p on oligonucleotides modeled after the native PYK1 UAS element but not when the Rap1p- and Gcr1p-binding sites were displaced by 5 nucleotides. This work demonstrates that the role of the Rap1p in the activation of glycolytic enzyme genes is to bind in their UAS elements and to facilitate the binding of Gcr1p at adjacent binding sites.

Base Sequence↗

PubMed2003

HoxA10 represses gene transcription in undifferentiated myeloid cells by interaction with histone deacetylase 2.

The homeodomain proteins, HoxA10 and Pbx1a, interact with negative cis elements to repress gene transcription in undifferentiated myeloid cells. The CYBB and NCF2 genes, which encode the gp91PHOX and p67PHOX proteins, are two such HoxA10-Pbx1a target genes. In previous studies, we found that HoxA10-Pbx1a represses transcription of these genes by two mechanisms: competition for DNA binding with transcriptional activators and endogenous repression activity. In these studies, we identify a novel molecular mechanism of endogenous transcriptional repression by HoxA10-Pbx1a. Endogenous repression activity of other Hox-Pbx1a complexes requires recruitment of transcriptional co-repressor proteins by Pbx1a. In contrast, our investigations have determined that HoxA10 has Pbx1a-independent endogenous repression activity. We find that this transcriptional repression activity is abrogated by histone deacetylase inhibitors, suggesting involvement of co-repressor proteins. Consistent with this, we identify HoxA10 amino acids 224-249 as a Pbx1-independent repression domain, which interacts with histone deacetylase 2. We have determined that this HoxA10 domain is not conserved with other Abd Hox proteins, although homology exists with other transcription factors and co-repressors. Understanding the roles different Hox proteins play in myeloid differentiation is a challenging problem. Our results suggest that insight into this problem can be obtained from biochemical characterization of the various molecular mechanisms of Hox protein function.

Blotting, Northern↗

PubMed1990

Plasmid pLS1-encoded RepA protein regulates transcription from repAB promoter by binding to a DNA sequence containing a 13-base pair symmetric element.

The repA gene product of the promiscuous plasmid pLS1 is a 45-amino acid repressor protein. The plasmid initiator of replication protein, RepB, is encoded by the repB gene which is situated downstream of repA. The results presented here demonstrate that both genes constitute a transcriptional unit. We show that the repA gene product inhibits transcription from the repAB promoter both in vitro and in vivo. By hydroxyl radical footprinting on both DNA strands, we show that RepA binds specifically to a plasmid region in which a 13-base pair element, showing a 2-fold rotational symmetry, is located. Within this symmetric element lies the -35 region of the repAB promoter. RepA binds into successive major grooves along one face of the DNA helix. The general architecture of RepA and of its interactions with DNA resembles that of the Cro repressor proteins of bacteriophages lambda and 434. We propose that RepA regulates the plasmid copy number by binding to its own promoter, thus controlling the synthesis of the plasmid initiator of replication protein.

Amino Acid Sequence↗

PubMed2000

The 1-kb-repeat-encoded DNA-binding protein as repressor of an alpha-glucosidase operon flanking the amplifiable sequence AUD1 of Streptomyces lividans.

High-copy-number amplification of the AUD1 element is frequently associated with the large chromosomal deletions responsible for genetic instability in Streptomyces lividans TK64. Five ORFs were found in a 7 kb region directly adjacent to AUD1. The putative products of ORF1, ORF2 and ORF3 showed similarities to ATP-binding cassette (ABC) sugar transporters, the deduced protein sequence of ORF4 displayed similarities to alpha-glucosidases whilst no homology to proteins with known functions was found for ORF5. ORF4 (renamed aglA) was expressed in Escherichia coli and the protein purified and characterized. An alpha-glucosidase activity was detected using the synthetic alpha-glucoside p-nitrophenyl alpha-D-glucopyranoside. Of the many oligosaccharides tested, only sucrose was hydrolysed at a measurable rate [specific activity 32.4 units (mg protein)(-1)] but no growth of S. lividans TK64 on sucrose was observed. A strain in which aglA was disrupted showed the same low alpha-glucosidase activity as strain TK64 and in both strains no stimulation of activity was seen by sucrose, trehalose or maltose; dextrin increased alpha-glucosidase activity about 10-fold. This probably resulted from induction of a second alpha-glucosidase-encoding gene. The AUD1 element contains three 1 kb repeats which encode DNA-binding proteins necessary for high-frequency amplification. In strains with a unique 1 kb repeat, disruption of the repeat led to a significant increase in the alpha-glucosidase activity. These results strongly suggest that the 1-kb-repeat-encoded proteins of AUD1 have a dual function: they are the repressors of the agl genes and they promote amplification of AUD1.

ATP-Binding Cassette Transporters↗

PubMed1983

Studies on the interaction of Trp holorepressor with several operators. Evidence that the target need not be palindromic.

The interaction of Trp repressor protein with partial trp operators was studied in vitro and in vivo. At high ratios of protein to DNA, Trp holorepressor formed stable complexes with DNA molecules containing half operators. When plasmids conferring the capacity to hyperproduce Trp repressor were present in trpOc strains of Escherichia coli, repression of downstream tryptophan synthase occurred. Palindromicity of the trp operator may facilitate stable interaction with Trp repressor, but this attribute need not be regarded as a critically essential structural feature. Sufficient information for the recognition by Trp repressor protein of an appropriate target resides within a DNA sequence of approximately ten base-pairs.

Bacterial Proteins↗

PubMed1996

Hypertonic activation and recovery of system A amino acid transport in renal MDCK cells.

Amino compounds are abundant within the renal inner medulla, but their possible role during hypertonic stress is not clear. Renal epithelial Madin-Darby canine kidney cells were used to examine the osmoregulation of system A transport, a major Na(+)-dependent process for neutral amino acid transport. System A activity was markedly increased after 6 h of hypertonic challenge, and intracellular alanine content increased more than twofold. The activation of system A was reversed after 24 h of hypertonic challenge. This downregulation was accompanied by the activation of betaine transport, as measured by gamma-aminobutyric acid uptake. Extracellular betaine prevented the early activation of system A. The hypertonic activation of system A was blocked by actinomycin D and cycloheximide. When cells were returned to isotonic medium after hypertonic activation, the recovery of system A transport also was partially inhibited by actinomycin D and puromycin. The results are consistent with the possibility that hypertonicity, by disrupting a repressor protein, leads to increased synthesis of a system A-related protein. The isotonic recovery may require synthesis of new repressor proteins.

Amino Acids↗

PubMed2000

Computer based analyses of the 5'-flanking regions of selected genes involved in the nucleotide excision repair complex.

We have previously observed that the mRNA of selected genes involved in nucleotide excision repair appear to be coordinately expressed in human tissues from patients with ovarian cancer, testicular cancer, malignant brain tumors, and other malignancies. Such genes include ERCC1, XPA, XPB, XPD, XPF, and XPG. Coordinate mRNA expression appears to be most impressive in non-malignant tissues. We therefore began to explore possible reasons why such coordinate expression should occur. DNA sequences for the above noted genes were obtained from GeneBank. Two different software programs were applied to the DNA sequence, to the area 5' to the start of exon I of each gene. Analyses were performed by computer. The length of the 5' area assessed, was based on previous reports that determined what portion of the genomic sequence comprised the 5' UTR of the promoter of the respective gene. Based on this approach, potential DNA binding sites for no less than three dozen proteins, were identified in the 5'-flanking region of each of the NER genes studied. For each gene, potential binding sites for activator proteins and for repressor proteins were identified. The 5'-flanking regions for each gene noted above, had binding sites in common for 14 proteins with transcription modulatory activity. Eleven of these proteins are known for activator activity; two are reported to have repressor activity, and one has both repressor and activator function. These data suggest a possible molecular basis for the previously observed coordinate mRNA expression of selected NER genes in human tissue specimens.

Adaptor Protein Complex alpha Subunits↗

PubMed2004

A role for the interdomain linker region of the Escherichia coli CytR regulator in repression complex formation.

Regulatory complexes formed by the CytR repressor protein and the cAMP receptor protein (CRP) prevent transcription initiation from several promoters in Escherichia coli. The formation of the complexes is mediated by protein-DNA interactions and protein-protein interactions between the two regulators. Interestingly, co-binding with CRP has a profound effect on the configuration of the DNA-binding targets preferred by CytR. When binding to DNA by itself, CytR binds preferentially to two octamer repeats in direct or inverted orientation, and separated by 2 bp. However, in the presence of CRP, CytR recognizes inverted repeats separated by 10-13 bp, or direct repeats separated by 1 bp. A fixed orientation of at least one CytR octamer repeat in close proximity to a CRP-binding target is a common architectural feature at promoters optimised for repression complex formation. These observations suggest that CRP alters the DNA-binding mode of CytR. Here, we have investigated the CRP-induced changes in CytR by protein footprinting and alanine-scanning mutagenesis. Our data suggest that a flexible interdomain linker region in CytR, connecting the DNA-binding domain to the dimerization domain allows the repressor protein to interact with DNA-binding sites in a highly relaxed manner, as shown previously, and plays an active role in transcription regulation. Thus, the interactions between CRP, CytR and DNA within the repression complex appear to be more extensive than anticipated. The results support and extend the view that the high degree of adaptability observed in the CytR/CRP regulatory system is obtained though multiple adjustable interactions between the implicated factors.

Amino Acid Sequence↗

PubMed2003

Target of rapamycin (TOR)-signaling and RAIP motifs play distinct roles in the mammalian TOR-dependent phosphorylation of initiation factor 4E-binding protein 1.

The translational repressor protein eIF4E-binding protein 1 (4E-BP1, also termed PHAS-I) is regulated by phosphorylation through the rapamycin-sensitive mTOR (mammalian target of rapamycin) pathway. Recent studies have identified two regulatory motifs in 4E-BP1, an mTOR-signaling (TOS) motif in the C terminus of 4E-BP1 and an RAIP motif (named after its sequence) in the N terminus. Other recent work has shown that the protein raptor binds to mTOR and 4E-BP1. We show that raptor binds to full-length 4E-BP1 or a C-terminal fragment containing the TOS motif but not to an N-terminal fragment containing the RAIP motif. Mutation of several residues within the TOS motif abrogates binding to raptor, indicating that the TOS motif is required for this interaction. 4E-BP1 undergoes phosphorylation at multiple sites in intact cells. The effects of removal or mutation of the RAIP and TOS motifs differ. The RAIP motif is absolutely required for phosphorylation of sites in the N and C termini of 4E-BP1, whereas the TOS motif primarily affects phosphorylation of Ser-64/65, Thr-69/70, and also the rapamycin-insensitive site Ser-101. Phosphorylation of N-terminal sites that are dependent upon the RAIP motif is sensitive to rapamycin. The RAIP motif thus promotes the mTOR-dependent phosphorylation of multiple sites in 4E-BP1 independently of the 4E-BP1/raptor interaction.

Adaptor Proteins, Signal Transducing↗

PubMed1984

Control of expression of the Tn10-encoded tetracycline resistance operon. II. Interaction of RNA polymerase and TET repressor with the tet operon regulatory region.

The promoter and operator sequences of the Tn10-encoded tetracycline resistance operon are determined in vitro by transcription studies of purified DNA restriction fragments, protection of guanosine from methylation by dimethylsulphate, and DNase I footprinting employing the purified TET repressor protein. In vitro transcription reveals three promoters with overlapping consensus sequences. Two of them, designated PR1 and PR2, are directed towards the tet repressor gene and the third, called PA, initiates transcription of the tet resistance gene. All three promoters are regulated simultaneously by the TET repressor protein, as demonstrated by in vitro transcription. Tetracycline functions as an inducer in these experiments. Two palindromic operator sequences in the tet operon control region, called O1 and O2, are occupied simultaneously by the TET repressor. Four guanosine residues in symmetric positions close to the centre of the palindromic operator sequences are protected from methylation in the repressor-operator complex. However, only one guanosine residue exhibits an enhanced reaction with dimethylsulphate under these conditions. Footprinting experiments reveal protection of phosphodiester bonds against DNase I slightly further than the palindromic sequence arrangement. Several phosphodiester bonds between the two operators are accessible for cleavage by DNase I in the repressor-operator complex. Two phosphodiester bonds within each operator sequence are cleaved by DNase I. This feature shows a clear assymmetry with the two inside cleavage positions of O1 and O2 being much less accessible for DNase I as compared to the two outside positions. A molecular mechanism of regulation of the Tn10-encoded tetracycline resistance operon is presented based on these and previous results.

Base Sequence↗

PubMed2002

Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation.

Although many co-activators have been identified for various nuclear receptors, relatively fewer co-repressors have been isolated and characterized. Here we report the identification of a novel testicular orphan nuclear receptor-4 (TR4)-associated protein (TRA16) that is mainly localized in the nucleus of cells as a repressor to suppress TR4-mediated transactivation. The suppression of TR4-mediated transactivation is selective because TRA16 shows only a slight influence on the transactivation of androgen receptor, glucocorticoid receptor, and progesterone receptor. Sequence analysis shows that TRA16 is a novel gene with 139 amino acids in an open reading frame with a molecular mass of 16 kDa, which did not match any published gene sequences. Mammalian two-hybrid system and co-immunoprecipitation assays both demonstrate that TRA16 can interact strongly with TR4. The electrophoretic mobility shift assay suggests that TRA16 may suppress TR4-mediated transactivation via decreased binding between the TR4 protein and the TR4 response element on the target gene(s). Furthermore, TRA16 can also block the interaction between TR4 and TR4 ligand-binding domain through interacting with TR4-DNA-binding and ligand-binding domains. These unique suppression mechanisms suggest that TRA16 may function as a novel repressor to selectively suppress the TR4-mediated transactivation.

Amino Acid Sequence↗

PubMed2002

Searching for DNA-protein interactions by lambda phage display.

We applied phage display technology to DNA-protein interaction studies. A cDNA expression library displayed on the surface of bacteriophage lambda was generated from the highly differentiated MMH E14 murine hepatic cell line. Selection of this library using the promoter sequence of the liver-enriched transcription factor HNF1alpha gene as ligate identified DNA-binding domains specifically interacting with different regions of this regulatory sequence. One of the selected phage showed 100% identity to a DNA-binding domain shared by differentiation specific element-binding protein, vasoactive intestinal peptide receptor-repressor protein and replication factor C and was further investigated. Specific binding of the selected protein domain was confirmed in a phage-independent context. By combining ELISA and South-Western assays using the selected phage and a bacterially expressed glutathione-S-transferase protein fused to the encoded DNA-binding domain, an array of multiple adjacent DNA-binding sites sharing a common consensus motif was identified. The strategy described represents a powerful tool to identify proteins that bind to DNA regulatory elements.

Adenosine Triphosphatases↗