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K Melcher

Publications and source records attributed to K Melcher.

12 recordsLinked to original sources
PubMed2004

New chemical crosslinking methods for the identification of transient protein-protein interactions with multiprotein complexes.

Most proteins function as multiprotein complexes or interact with multiprotein complexes. Identification of protein-protein interactions in the context of their physiologically relevant complexes is therefore key to fully understand the cellular machinery. Here I discuss advances in chemical crosslinking methods that allow investigators to map direct subunit contacts in transient interactions with multimeric complexes. Methods discussed fall into two categories: (i) in vitro approaches with localized, inducible crosslinking reagents and (ii) in vivo approaches with unlocalized crosslinkers.

Animals↗
PubMed2001

Gal80-Gal80 interaction on adjacent Gal4p binding sites is required for complete GAL gene repression.

Regulation of the GAL genes of Saccharomyces cerevisiae is determined by the interplay of the transcriptional activator Gal4p and the repressor Gal80p, which binds and masks the activation domain of Gal4p under non-inducing conditions. Here we demonstrate that Gal80p dimerizes with high affinity and that this dimerization appears to stabilize the Gal4p-Gal80p interaction and also, indirectly, the Gal4p-DNA interaction in a (Gal4p)2(Gal80p)2DNA complex. In addition, Gal80 dimers transiently interact with each other to form higher order multimers. We provide evidence that adjacent Gal4p binding sites, when correctly spaced, greatly stabilize Gal80p dimer-dimer interactions and that this stabilization results in the complete repression of GAL genes with multiple Gal4p binding sites. In contrast, GAL genes under the control of a single Gal4p binding site do not stabilize Gal80p multimers, resulting in significant and biologically important transcriptional leakage. Cooperative binding experiments indicate that Gal80p dimer-dimer interaction probably does not lead to a stronger Gal4p-Gal80p interaction, but most likely to a more complete shielding of the Gal4p activation domain.

Dimerization↗
PubMed2000

The strength of acidic activation domains correlates with their affinity for both transcriptional and non-transcriptional proteins.

Activation domains (ADs) appear to work by making specific protein-protein contacts with the transcriptional machinery. However, ADs show no apparent sequence conservation, they can be functionally replaced by a number of random peptides and unrelated proteins, and their function does not depend on sustaining a complex tertiary structure. To gain a broader perspective on the nature of interactions between acidic ADs and several of their proposed targets, the in vivo strengths of viral, human, yeast, and artificial activation domains were determined under physiological conditions, and mutant ADs with increased in vivo potencies were selected. The affinities between ADs and proposed targets were determined in vitro and all interactions were found to be of low-level affinity with dissociation constants above 10(-7)M. However, in vivo potencies of all ADs correlated nearly perfectly with their affinities for transcriptional proteins. Surprisingly, the weak interactions of the different ADs with at least two non-transcriptional proteins show the same rank order of binding and AD mutants selected for increased in vivo strength also have increased affinities to non-transcriptional proteins. Based on these results, isolated acidic ADs can bind with relatively low-level specificity and affinity to many different proteins and the strength of these semi-specific interactions determine the strength of an AD. I suggest that ADs expose flexible hydrophobic elements in an aqueous environment to contact hydrophobic patches over short distances, shifting specificity of activators largely to the DNA colocalization of arrays of ADs and targets.

Adenosine Triphosphatases↗
PubMed2000

Zero background yeast reporter plasmids.

UAS-less reporter plasmids are widespread and powerful tools for the identification and analysis of binding sites for transcriptional activators. The common reporter plasmids for the yeast Saccharomyces cerevisiae are multicopy (2mu) vectors with the CYC1 core promoter upstream of the lacZ gene. Insertion of putative or known activator binding sites upstream of the core promoter puts lacZ (beta-galactosidase) expression under the control of the corresponding activator. Although these constructs have proved to work well for most purposes, they have certain limitations: (1) they give significant and carbon-source-dependent lacZ background expression; (2) unlike most other yeast promoters, the CYC1 upstream region has a partially open chromatin structure with an accessible TATA box; (3) they use only a single, moderately sensitive reporter; and (4) the use of multicopy vectors can result in activator titration. Here, we introduce novel reporter plasmids based on the yeast MEL1 (alpha-galactosidase) gene that can overcome all of these limitations. It is also shown that background expression is due to fortuitous activator binding sites within the plasmid backbones that are insufficiently shielded from the core promoters in the common CYC1 reporter plasmids.

Binding Sites↗
PubMed2000

A modular set of prokaryotic and eukaryotic expression vectors.

A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine-GST) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (TEV) protease, (iii) PCR cloning design that results in a product of proteolytic cleavage with only one (a single glycine) or two (gly-ala) amino acids at the N-terminus of the protein, and (iv) expression in either Escherichia coli or Saccharomyces cerevisiae. In addition, singly hexahistidine-tagged proteins can be produced for purification under denaturing conditions and some vectors allow addition of five amino acid kinase recognition sites for easy radiolabeling of proteins. To illustrate the use of these vectors, all regulatory components of the yeast GAL regulon, rather than abundant highly soluble proteins, were produced and purified under native or denaturing conditions, and their biological activity was confirmed.

Amino Acid Sequence↗
PubMed1998

Evidence for two modes of cooperative DNA binding in vivo that do not involve direct protein-protein interactions.

BACKGROUND: The promoter regions of most eukaryotic genes contain binding sites for more than one transcriptional activator and these activators often bind cooperatively to promoters. The most common type of cooperativity is supported by direct protein-protein interactions. Recent studies have shown that proteins that do not specifically interact with one another can bind cooperatively to chromatin in vitro. probably by the localized destabilization of nucleosome structure by one factor, facilitating binding of another to a nearby site. This mechanism does not require that the transcription factors have activation domains. We have examined whether this phenomenon occurs in vivo. RESULTS: Unrelated non-interacting proteins can bind DNA cooperatively in yeast cells; this cooperative binding can contribute significantly to transcriptional activation, does not require that both factors have activation domains and is only operative over relatively short distances. In addition to this 'short-range' mechanism, unrelated non-interacting proteins can bind cooperatively to sites separated by hundreds of base pairs, so long as both have potent activation domains. CONCLUSION: Cooperative binding of transcription factors in vivo can occur by several mechanisms, some of which do not require direct protein-protein interactions and which cannot be detected in vitro using naked DNA templates. These findings must be taken into account when evaluating mechanisms for synergistic transcriptional activation.

Bacterial Proteins↗
PubMed1996

New chemistry for the study of multiprotein complexes: the six-histidine tag as a receptor for a protein crosslinking reagent.

BACKGROUND: To study very large macromolecular complexes, it would be useful to be able to incorporate probe molecules, such as fluorescent tags or photoactivatable crosslinkers, into specific sites on proteins. Current methods for doing this use relatively large amounts of highly purified protein, limiting the general utility of these approaches. The need for covalent posttranslational chemistry also makes it extremely difficult to use modified proteins in studies of native complexes in crude lysates or in living cells. We set out to develop a protein tag that would circumvent these problems. RESULTS: A very simple type of molecular recognition, metal-ligand complexation, can be used to deliver a nickel-based crosslinking reagent to proteins containing a six-histidine (His6) tag. When activated with a peracid, the His6-Ni complex mediates oxidative crosslinking of nearby proteins. The crosslinking reaction does not involve freely diffusible intermediates, and thus only those proteins in close proximity to the His6-tagged polypeptide are crosslinked. CONCLUSIONS: The His6 tag, commonly used as an affinity handle for the purification of recombinant proteins, can also be used as an internal receptor for an oxidative protein-crosslinking reagent. No covalent protein modifications are necessary, since the His6 tag is introduced at the DNA level. The crosslinking reaction is fast, efficient in most cases, and provides products that are easily separated from most other proteins present. This methodology should find widespread use in the study of multiprotein complexes.

Binding Sites↗
PubMed1995

A highly conserved ATPase protein as a mediator between acidic activation domains and the TATA-binding protein.

Biochemical and genetic studies suggest the existence of mediators that work between the activation domains (ADs) of regulatory proteins and the basic transcriptional machinery. We have previously shown genetically that Sug1 interacts with the AD of the yeast activator Ga14. Here we provide evidence that the Sug1 protein of yeast binds directly to the ADs of Ga14 and the viral activator, VP16. Sug1 protein is associated with the TATA-binding protein in vivo and binds to it in vitro, consistent with a mediator function. We also demonstrate that Sug1 is not a component of the 26S proteasome, contrary to previous reports. Sug1 is a member of a large, highly conserved family of ATPases, implying a role for ATP hydrolysis in the activation of transcription.

Adenosine Triphosphatases↗
PubMed1995

Molecular analysis of the yeast SER1 gene encoding 3-phosphoserine aminotransferase: regulation by general control and serine repression.

Although serine and glycine are ubiquitous amino acids the genetic and biochemical regulation of their synthesis has not been studied in detail. The SER1 gene encodes 3-phosphoserine aminotransferase which catalyzes the formation of phosphoserine from 3-phosphohydroxy-pyruvate, which is obtained by oxidation of 3-phosphoglycerate, an intermediate of glycolysis. Saccharomyces cerevisiae cells provided with fermentable carbon sources mainly use this pathway (glycolytic pathway) to synthesize serine and glycine. We report the isolation of the SER1 gene by complementation and the disruption of the chromosomal locus. Sequence analysis revealed an open reading frame encoding a protein with a predicted molecular weight of 43,401 Da. A previously described mammalian progesterone-induced protein shares 47% similarity with SER1 over the entire protein, indicating a common function for both proteins. We demonstrate that SER1 transcription is regulated by the general control of amino-acid biosynthesis mediated by GCN4. Additionally, DNaseI protection experiments proved the binding of GCN4 protein to the SER1 promoter in vitro and three GCN4 recognition elements (GCREs) were identified. Furthermore, there is evidence for an additional regulation by serine end product repression.

Amino Acid Sequence↗
PubMed1995

GAL4 interacts with TATA-binding protein and coactivators.

A major goal in understanding eukaryotic gene regulation is to identify the target(s) of transcriptional activators. Efforts to date have pointed to various candidates. Here we show that a 34-amino-acid peptide from the carboxy terminus of GAL4 is a strong activation domain (AD) and retains at least four proteins from a crude extract: the negative regulator GAL80, the TATA-binding protein (TBP), and the putative coactivators SUG1 and ADA2. TFIIB was not retained. Concentrating on TBP, we demonstrate in in vitro binding assays that its interaction with the AD is specific, direct, and salt stable up to at least 1.6 M NaCl. The effects of mutations in the GAL4 AD on transcriptional activation in vivo correlate with their affinities to TBP. A point mutation (L114K) in yeast TBP, which has been shown to compromise the mutant protein in both binding to the VP16 AD domain and activated transcription in vitro, reduces the affinity to the GAL4 AD to the same degree as to the VP16 AD. This suggests that these two prototypic activators make similar contacts with TBP.

Amino Acid Sequence↗
PubMed1992

Genetic analysis of serine biosynthesis and glucose repression in yeast.

Serine and glycine biosynthesis in yeast proceed by two pathways: a "glycolytic" pathway, using 3-phosphoglycerate, and a "gluconeogenic" pathway, using glyoxylate. We used a mutation in the cat1 gene to abolish the glucose-repressible "gluconeogenic" pathway and re-isolated two mutants, ser1 and ser2, in the "glycolytic" pathway. The ser1 mutation corresponded to phosphoserine transaminase and ser2 to that of phosphoserine phosphatase. Mutagenesis of a ser1 ser2 cat1 triple mutant facilitated the isolation of a mutation in a new gene, SER10. SER10 appears to be part of a pathway which, under normal growth conditions, is less important in serine biosynthesis. The ser1 ser2 ser10 triple mutants were totally serine auxotrophic on glucose media but serine prototrophic during growth on non-fermentable carbon sources. This phenotype was used to select for possible regulatory mutants that synthesize serine by the gluconeogenic pathway even in the presence of glucose, e.g., with a non-glucose repressible glyoxylate cycle. In an alternative approach to isolate such mutants URA3 and TRP1 expression were placed under the control of the glucose-repressible FBP1 (fructose-1,6-bisphosphatase) promoter. Although both systems resulted in strong selection pressure we could not isolate constitutively derepressed mutants. These results indicate that transcription of glucose-repressible gluconeogenic enzymes is mainly dependent on positive regulatory elements.

Base Sequence↗