Biomedical subjects

N Lehming

Publications and source records attributed to N Lehming.

17 recordsLinked to original sources

PubMed2001

Why Ppr1p is a weak activator of transcription.

Upon uracil depletion, the transcriptional activator Ppr1p stimulates expression of the Saccharomyces cerevisiae URA3 gene only four-fold. We performed a split-ubiquitin screen with Tup1p as bait, and we found that the global repressor Tup1p interacts with the transcriptional activator Ppr1p both in vivo and in vitro. The interaction is biologically significant, since the deletion of the TUP1 gene as well as the removal of the Tup1p-binding domain from Ppr1p results in an increased expression of the URA3 gene. Our results suggest that Tup1p blocks Ppr1p directly, and that Ppr1p is a weak activator of transcription because of its interaction with Tup1p. Thus we were able to demonstrate that the global repressor Tup1p can modulate transcription by interacting with an activator.

DNA-Binding Proteins↗

PubMed2001

Genome-wide expression analysis of a Saccharomyces cerevisiae strain deleted for the Tup1p-interacting protein Cdc73p.

Tuplp is a general corepressor in Saccharomyces cerevisiae. We performed a split-ubiquitin screen with Tup1p as bait, in order to understand how Tuplp mediates repression. Cdc73p was the only component of a holoenzyme of transcription isolated as a Tuplp-interacting protein in this screen. Expression analysis of a strain deleted for CDC73 on a genome-wide scale revealed that Cdc73p is involved in the positive and negative regulation of transcription. Our results show that a subset of genes is regulated by both Cdc73p and Tup1p. Furthermore, our results indicate that Cdc73p plays a role in the repression of telomere-proximal genes, which are not repressed by Tup1p.

Fungal Proteins↗

PubMed2000

Srb7p is a physical and physiological target of Tup1p.

The holoenzyme of transcription integrates the positive and negative signals from the promoters of eukaryotic genes. We demonstrate that the essential holoenzyme component Srb7p is a physiologically relevant target of the global repressor Tup1p in Saccharomyces cerevisiae. Tup1p binds Srb7p in vivo and in vitro, and all genes tested that are repressed by Tup1p are derepressed when wild-type Srb7p is replaced by a mutant derivative of Srb7p that is no longer capable of interacting with Tup1p. Therefore, Srb7p is the first holoenzyme component essential for repression by Tup1p for which a physical interaction with Tup1p has been demonstrated. Furthermore, we find that Srb7p also binds Med6p and that this interaction is necessary for full transcriptional activation by different activators. Our finding that Med6p and Tup1p compete for the interaction with Srb7p suggests a model for Tup1p-mediated repression.

Amino Acid Sequence↗

PubMed2000

A new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter.

The split-ubiquitin assay detects protein interactions in vivo. To identify proteins interacting with Gal4p and Tup1p, two transcriptional regulators, we converted the split-ubiquitin assay into a generally applicable screen for binding partners of specific proteins in vivo. A library of genomic Saccharomyces cerevisiae DNA fragments fused to the N-terminal half of ubiquitin was constructed and transformed into yeast strains carrying either Gal4p or Tup1p as a bait. Both proteins were C-terminally extended by the C-terminal half of ubiquitin followed by a modified Ura3p with an arginine in position 1, a destabilizing residue in the N-end rule pathway. The bait fusion protein alone is stable and enzymatically active. However, upon interaction with its prey, a native-like ubiquitin is reconstituted. RUra3p is then cleaved off by the ubiquitin-specific proteases and rapidly degraded by the N-end rule pathway. In both screens, Nhp6B was identified as a protein in close proximity to Gal4p as well as to Tup1p. Direct interaction between either protein and Nhp6B was confirmed by coprecipitation assays. Genetic analysis revealed that Nhp6B, a member of the HMG1 family of DNA-binding proteins, can influence transcriptional activation as well as repression at a specific locus in the chromosome of the yeast S. cerevisiae.

Base Sequence↗

PubMed2000

Srb7p is essential for the activation of a subset of genes.

The mediator complex in the RNA polymerase II holoenzyme is known to be involved in transcriptional activation. The role of the essential mediator component Srb7p has been difficult to investigate, since no conditional lethal allele has been available to date. While the expression of Srb7p under the control of a repressible promoter is not sufficient to reduce the level of Srb7p beneath the threshold for survival, we have been able to isolate a clone termed ts16 which confers a temperature sensitive phenotype. ts16 contains an insertion mutation that requires translational frameshifting for correct expression of Srb7p, leading to extremely low protein levels. Strains bearing the ts16 construct show mild defects in the transcription of constitutive genes like TDH1 but severely affect activated transcription, e.g. of the GAL1 gene. In contrast, CUP1, which is also independent of other holoenzyme components, is not affected by ts16.

Alleles↗

PubMed2000

A new method for the selection of protein interactions in mammalian cells.

In the present study we present a new method that allows for the selection of protein interactions in mammalian cells. We have used this system to verify two interactions previously characterized in vitro. (1) The interaction between human TATA-binding protein 1 and nuclear factor kappaB and (2) the association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1alpha, a protein implicated in chromatin remodelling. We observe for the first time that these interactions also occur in vivo. One protein was fused to the N-terminal half of ubiquitin, while the interacting partner was fused to the C-terminal half of ubiquitin, that was itself linked to guanine phosphoryltransferase 2 (gpt2) modified to begin with an arginine residue. Upon interaction of both proteins, ubiquitin is reconstituted, and its association with the Rgpt2 reporter is subsequently cleaved off by ubiquitin-processing enzymes. The presence of arginine in the Rgpt2 gene product leads to the degradation of the product by the N-end rule pathway. In the human fibroblast cell line HT1080HPRT(-) (that is deficient in the enzyme for hypoxanthine-guanine phosphoribosyltransferase) cells in which interaction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-thioguanine medium. This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable.

Antigens, Nuclear↗

PubMed1999

Analysis of the in vivo interaction between a basic repressor and an acidic activator.

The artificial basic repressor SSB24 represses transcription of a reporter construct activated by GCN4. We show that the positively charged SSB24 and the negatively charged acidic activator GCN4 interact in vitro and in vivo. However, deleting the interaction domain from the GCN4 activator does not result in loss of repression by SSB24. Similarly, transcription activated by the holoenzyme component SRB2 is repressed, although SSB24 and SRB2 do not interact. Repression by SSB24 therefore does not depend on the observed protein-protein interaction between SSB24 and GCN4.

Acids↗

PubMed1998

Chromatin components as part of a putative transcriptional repressing complex.

The Drosophila HMG1-like protein DSP1 was identified by its ability to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion in yeast. We show here that DSP1 as well as its mammalian homolog hHMG2 bind to the mammalian protein SP100B and that SP100B in turn binds to human homologs of HP1. The latter is a Drosophila protein that is involved in transcriptional silencing. Each of these proteins represses transcription when tethered to DNA in mammalian cells. These results suggest how heterochromatin proteins might be recruited to specific sites on DNA with resultant specific effects on gene expression.

Animals↗

PubMed1995

Interactions of a Rel protein with its inhibitor.

Cactus, a Drosophila homologue of I kappa B, binds to and inhibits Dorsal, a homologue of the p50 and p65 components of NF-kappa B. We describe experiments in yeast with various Dorsal and Cactus derivatives showing that Cactus blocks the DNA binding and nuclear localization functions of Dorsal. In contrast, Dorsal's transcriptional activating region is functional in the Dorsal-Cactus complex. We identify two Dorsal mutants, Dorsal C233R and Dorsal S234P, that escape Cactus inhibition in vivo, and we show that these mutants fail to interact with Cactus in vitro. From this and data of others, we identify the likely surface of Dorsal that binds Cactus. We also describe a modified PCR mutagenesis procedure, easier to use than conventional methods, that produces a library of high complexity.

Amino Acid Sequence↗

PubMed1994

An HMG-like protein that can switch a transcriptional activator to a repressor.

One protein can activate some genes and repress others in the same cell. The Drosophila protein Dorsal (which, like the human protein NF-kappa B3, is a member of the Rel family of transcriptional activators) activates the twist gene and represses the zen gene in the ventral region of early embryos. Here we describe a Drosophila HMG1 protein, called DSP1 (dorsal switch protein), that converts Dorsal and NF-kappa B from transcriptional activators to repressors. This effect requires a sequence termed a negative regulatory element (NRE), found adjacent to Dorsal-binding sites in the zen promoter and adjacent to the NF-kappa B-binding site in the human interferon-beta (IFN-beta) enhancer. Previous studies have shown that another type of HMG protein, HMG I(Y), can stimulate NF-kappa B activity. Thus, the HMG-like proteins DSP1 and HMG I(Y) can determine whether a specific regulator functions as an activator or a repressor of transcription.

Amino Acid Sequence↗

PubMed1993

New eukaryotic transcriptional repressors.

Transcriptional activating sequences have been described that are encoded by parts of the genome of Escherichia coli. These acidic peptides, fused to a DNA-binding fragment of the yeast transcriptional activator GAL4, activate transcription of a gene in a wide array of eukaryotes, provided that gene bears GAL4-binding sites nearby. Here we describe an E. coli-encoded sequence that, when attached to the same DNA-binding fragment (GAL4(1-147)), converts that fragment into a repressor. Thus, as assayed in yeast or in vitro in yeast extracts, this molecule represses transcription when bound upstream of a variety of different activators. Two additional repressing regions that work when tethered upstream, a multiple mutant derivative of the original isolate and a synthetic peptide are, like the original isolate, highly basic. At least one activator can be inhibited by the mutant but not by the parental repressing region. These and other findings suggest that these repressing regions interact with and inhibit the activity of activating regions bound nearby on DNA.

Amino Acid Sequence↗

PubMed1991

A model of the lac repressor-operator complex based on physical and genetic data.

Computer graphics were used to build a molecular model of the complex of Lac repressor and lac operator. The model is based (a) on the NMR data of the Kaptein group [Boelens, R., Lamerichs, R. M. J. N., Rullmann, J. A. C., van Boom, J. H. & Kaptein, R. (1988) Protein Sequence Data Anal. 1, 487-498] and (b) on our genetic and biochemical data including specificity changes [Lehming, N., Sartorius, J., Kisters-Woike, B., von Wilcken-Bergmann, B. & Müller-Hill, B. (1990) EMBO J. 9, 615-621]. Effects of amino acid exchanges in the recognition helix could be predicted by the model and were subsequently tested and confirmed by genetic experiments. Comparison of the modelled lac complex with the known crystallographic structures of several helix-turn-helix DNA complexes reveals striking similarities and suggests rules which govern the recognition between particular amino acid side chains and particular base pairs in these systems.

Amino Acid Sequence↗

PubMed1991

The roles of residues 5 and 9 of the recognition helix of Lac repressor in lac operator binding.

We constructed expression libraries for Lac repressor mutants with amino acid exchanges in positions 1, 2, 5 and 9 of the recognition helix. We then analysed the interactions of residues 5 and 9 with operator variants bearing single or multiple symmetric base-pair exchanges in positions 3, 4 and 5 of the ideal fully symmetric lac operator. We isolated 37 independent Lac repressor mutants with five different amino acids in position 5 of the recognition helix that exhibit a strong preference for particular residues in position 2 and, to a lesser extent, in position 1 of the recognition helix. Our results suggest that residue 5 of the recognition helix (serine 21) contributes to the specific recognition of base-pair 4 of the lac operator. They further suggest that residue 9 of the recognition helix (asparagine 25) interacts non-specifically with a phosphate of the DNA backbone, possibly between base-pairs 2 and 3.

Amino Acid Sequence↗

PubMed1990

Mutant lac repressors with new specificities hint at rules for protein--DNA recognition.

Proteins which recognize specific sequences of DNA play a fundamental role in the regulation of protein synthesis in all organisms. A particular helix of the bacterial protein lac repressor recognizes the bases in the major groove of the lac operator. We show that the first two residues of this recognition helix interact independently with two base pairs. This allows us in many cases to predict repression as an indicator of strength of the repressor-operator complex. Rules of recognition can be derived for 16 symmetric operators. They also apply to the gal repressor and possibly to other bacterial repressors.

Amino Acid Sequence↗

PubMed1989

lac repressor mutants with double or triple exchanges in the recognition helix bind specifically to lac operator variants with multiple exchanges.

Several lac repressor mutants have been isolated which repress beta-galactosidase synthesis in Escherichia coli up to 200-fold. They do so by binding specifically to particular symmetrical lac Oc operator variants. The mutations in the lac repressor are localized in two separate parts of the recognition helix comprising (i) residues 1 and 2 which interact with base pairs 4 and 5 of lac operator and (ii) residue 6 which recognizes operator base pair 6. Mutations of residues 1 and 2 may be combined with a mutation of residue 6. The resulting mutant protein binds specifically to an operator variant with three symmetric exchanges in base pairs 4, 5 and 6.

Base Sequence↗

PubMed1988

Recognition helices of lac and lambda repressor are oriented in opposite directions and recognize similar DNA sequences.

Exchanges in positions 1 and 2 of the putative recognition helix allow lac repressor to bind to ideal lac operator variants in which base pair 4 has been replaced. We show here that an Arg-22----Asn exchange in position 6 of the putative recognition helix of lac repressor abolishes lac repressor binding to ideal lac operator. This lac repressor variant, however, binds to a variant of the ideal lac operator 5' TTTGAGCGCTCAAA 3' in which the original G.C of position 6 has been replaced by T.A. This result and our previous data confirm our suggestion that the N terminus of the recognition helix of lac repressor enters the major groove close to the center of symmetry of lac operator and that its C terminus leaves the major groove further away from the center of symmetry. The consequences of this model are discussed in regard to various phage and bacterial repressor operator systems.

Base Sequence↗

PubMed1987

The interaction of the recognition helix of lac repressor with lac operator.

We have constructed a system which allows systematic testing of repressor--operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor.

Amino Acid Sequence↗