Biomedical subjects

A K Vershon

Publications and source records attributed to A K Vershon.

32 records · Page 2Linked to original sources

PubMed1995

A homeo domain protein lacking specific side chains of helix 3 can still bind DNA and direct transcriptional repression.

A series of mutations in the homeo domain of the yeast alpha 2 protein were constructed to test, both in vivo and in vitro, predictions based on the alpha 2-DNA cocrystal structure described by Wolberger et al. (1991). The effects of the mutations were observed in three different contexts using authentic target DNA sequences: alpha 2 binding alone to specific DNA, alpha 2 binding cooperatively with MCM1 to specific DNA, and alpha 2 binding cooperatively with a1 to specific DNA. As expected, changes in the amino acid residues that contact DNA in the X-ray structure severely compromised the ability of alpha 2 to bind DNA alone and to bind DNA cooperatively with MCM1. In contrast, many of these same mutations, including a triple change that altered all the "recognition" residues of helix 3, had little or no effect on the cooperative binding of alpha 2 and a1 to specific DNA, as determined both in vivo and in vitro. These results show that the ability of a homeo domain protein to correctly select and repress target genes does not necessarily depend on the residues commonly implicated in sequence-specific DNA binding.

Amino Acid Sequence↗

PubMed1993

A short, disordered protein region mediates interactions between the homeodomain of the yeast alpha 2 protein and the MCM1 protein.

Homeodomains are folded into a characteristic three-dimensional structure capable of recognizing DNA in a sequence-specific manner. We show that correct target site selection by the yeast alpha 2 protein requires, as well as its homeodomain, an adjacent short and apparently unstructured region of the protein. This flexible homeodomain extension is responsible for specifying an interaction with a second regulatory protein, MCM1, which permits the cooperative binding of the two proteins to an operator. Two additional experiments suggest that this extension-homeodomain arrangement is likely to have some generality. First, when the extension of alpha 2 is grafted onto the Drosophila engrailed homeodomain, it yields a protein with the DNA binding specificity of engrailed and the ability to bind cooperatively to DNA with MCM1. Second, the alpha 2 extension specifies interaction not only with the yeast MCM1 protein, but also with the related human protein SRF.

Amino Acid Sequence↗

PubMed1992

Meiotic induction of the yeast HOP1 gene is controlled by positive and negative regulatory sites.

The process of meiosis and sporulation in the yeast Saccharomyces cerevisiae is a highly regulated developmental pathway dependent on genetic as well as nutritional signals. The HOP1 gene, which encodes a component of meiotic chromosomes, is not expressed in mitotically growing cells, but its transcription is induced shortly after yeast cells enter the meiotic pathway. Through a series of deletions and mutations in the HOP1 promoter, we located two regulatory sites that are essential for proper regulation of HOP1. One site, called URS1H, brings about repression of HOP1 in mitotic cells and functions as an activator sequence in cells undergoing meiosis. The second site, which we designated UASH, acts as an activator sequence in meiotic cells and has similarity to the binding site of the mammalian CCAAT/enhancer binding protein (C/EBP). Both sites are required for full meiotic induction of the HOP1 promoter. We conclude that in mitotic yeast cells, the URS1H site maintains the repressed state of the HOP1 promoter, masking the effect of the UASH site. Upon entry into meiosis, repression is lifted, allowing the URS1H and UASH sites to activate high-level transcription.

Base Sequence↗

PubMed1991

Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions.

The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast. We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence. The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm. Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains. Moreover, the orientation of the recognition helix on the DNA is also conserved. This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed. Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.

Base Sequence↗

PubMed1991

Crystallization and preliminary X-ray diffraction studies of a MAT alpha 2-DNA complex.

Crystals have been obtained of the DNA-binding domain of the yeast MAT alpha 2 repressor bound to a 21 base-pair DNA site. The crystals are grown from polyethylene glycol and CaCl2 and form in space group P2(1) with a = 60.1 A, b = 39.4 A, c = 68.7 A and beta = 98 degrees. They diffract to 2.9 A resolution and contain one protein-DNA complex in the crystallographic asymmetric unit.

Base Sequence↗

PubMed1991

Secondary structure of the homeo domain of yeast alpha 2 repressor determined by NMR spectroscopy.

The yeast alpha 2 protein is a regulator of cell type in Saccharomyces cerevisiae. It represses transcription of a set of target genes by binding to an operator located upstream of each of these genes. The alpha 2 protein shares weak sequence similarity with members of the homeo domain family; the homeo domain is a 60-amino-acid segment found in many eukaryotic transcriptional regulators. In this paper we address the question of whether alpha 2 is structurally related to prototypical members of the homeo domain family. We used solution 1H and 15N nuclear magnetic resonance [NMR] spectroscopy to determine the secondary structure of an 83-amino-acid residue fragment of alpha 2 that contains the homeo domain homology. We have obtained resonance assignments for the backbone protons and nitrogens of the entire 60-residue region of the putative homeo domain and for most of the remainder of the alpha 2 fragment. The secondary structure was determined by using NOE connectivities between backbone protons, 3JHN-H alpha coupling constants, and dynamical information from the hydrogen exchange kinetics of the backbone amides. Three helical segments exist in the alpha 2 fragment consisting of residues 11-23, 32-42, and 46-60 (corresponding to residues 138-150, 159-169, and 173-187 of the intact protein). The positions of these three helices correspond extremely well to those of the Drosophila Antennapedia (Antp) and engrailed (en) homeo domains, whose three-dimensional structures have recently been determined by NMR spectroscopy and X-ray crystallography, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

NMR studies of Arc repressor mutants: proton assignments, secondary structure, and long-range contacts for the thermostable proline-8----leucine variant of Arc.

Arc repressor is a 53-residue sequence-specific DNA binding protein. We report the assignment of the proton NMR spectrum and the secondary structure for the thermostable PL8 variant of Arc. This mutant, which differs from wild type by a Pro-8----Leu substitution, was chosen for study because its enhanced stability allows spectra to be acquired at elevated temperatures where spectral resolution is higher. The first five residues of the protein play important roles in DNA binding but appear to be disordered in solution. Residues 6-14 form the remaining part of the N-terminal DNA binding region of the protein and assume an antiparallel beta-conformation. This indicates that Arc is a member of a new class of DNA binding proteins. The observed interresidue nuclear Overhauser effects are consistent with a beta-strand, gamma-turn, beta-strand structure for the residue 6-14 region, although other structures are also consistent with the data. The remaining portion of the protein is predominantly alpha-helical. Residues 16-26 and 35-50 form amphipathic alpha-helices which may pack together in a four-helix bundle in the protein dimer.

Amino Acid Sequence↗

PubMed1989

The Arc and Mnt repressors. A new class of sequence-specific DNA-binding protein.

Genetic, biochemical, and biophysical studies have begun to reveal details of the structures of Arc and Mnt and show that these repressors use residues at their N-terminal ends for operator recognition and binding. Some of the DNA contacts made by these residues have been identified, and this information together with NMR studies has permitted the construction of models of the DNA binding region. Although the accuracy of these models remains to be determined, it seems clear that Arc and Mnt are members of a new class of DNA-binding proteins.

Amino Acid Sequence↗

PubMed1989

Sequence-specific binding of arc repressor to DNA. Effects of operator mutations and modifications.

A set of arc operators with transition and/or transversion mutations at each operator base pair has been constructed. By determining the ability of Arc to bind these variant operators, the importance of each base pair for Arc recognition has been assessed. Methylation protection experiments have also been used to probe points of close contact between Arc and most of the mutant operators. These data, together with phosphate interference results obtained previously for the wild type operator, provide information about the operator surface that is contacted when Arc binds.

Bacteriophages↗

PubMed1987

Bacteriophage P22 Mnt repressor. DNA binding and effects on transcription in vitro.

We have examined the binding of Mnt repressor to operator DNA in vitro and have determined how this binding affects the level of transcription from two nearby promoters, Pant and Pmnt. Mnt binds to a region of DNA that overlaps the startpoint of transcription of Pant and the -35 region of Pmnt. Mnt represses transcription in vitro from Pant and enhances transcription from Pmnt. Protection and interference experiments show that Mnt binds to a single, 17 base-pair operator site. The operator sequence and the protein-DNA contacts are symmetric. Mnt makes major groove contacts on both faces of the operator DNA. At pH 7.5, 200 mM-KCl, 22 degrees C, the Mnt tetramer binds operator with high affinity (Kd = 2.2 X 10(-11M) and the protein-DNA complex is quite stable (t1/2 = 48 min). Operator binding shows large dependencies on pH, salt concentration, and temperature.

Base Sequence↗

PubMed1987

Interaction of the bacteriophage P22 Arc repressor with operator DNA.

Are repressor binds to a single, partially symmetric, 21 base-pair operator site that is centered between the -10 and -35 regions of the Pant promoter. Protection and interference experiments show that Arc makes contacts with the operator on one side of the DNA helix. Although Arc is a small protein (53 residues/subunit), it makes contacts that are farther from the center of the operator than those made by many larger repressors. These extended contacts include the phosphate groups at the ends of the 21 base-pair site. Under standard conditions (pH 7.5, 100 mM-KCl, 3 mM-MgCl2, 22 degrees C) half-maximal operator binding is observed at an Arc concentration of 2.5 X 10(-9) M and the protein-DNA complex is very stable (t1/2 approximately equal to 80 min).

DNA, Viral↗

PubMed1986

Isolation and analysis of arc repressor mutants: evidence for an unusual mechanism of DNA binding.

We have isolated 64 different missense mutations at 36 out of 53 residue positions in the Arc repressor of bacteriophage P22. Many of the mutant proteins with substitutions in the C-terminal 40 residues of Arc have reduced intracellular levels and probably have altered structures or stabilities. Mutations in the N-terminal ten residues of Arc cause large decreases in operator DNA binding affinity without affecting the ability of Arc to fold into a stable three-dimensional structure. We argue that these N-terminal residues are important for operator recognition but that they are not part of a conventional helix-turn-helix DNA binding structure. These results suggest that Arc may use a new mechanism for sequence specific DNA binding.

Amino Acid Sequence↗

PubMed1985

The bacteriophage P22 arc and mnt repressors. Overproduction, purification, and properties.

The arc and mnt genes of bacteriophage P22 encode small repressor proteins. We have cloned these genes onto plasmids that overproduce Arc and Mnt to greater than 1% of the soluble cellular protein. Both proteins were purified to greater than 95% homogeneity, and N-terminal sequences and amino acid compositions were determined. These data, in combination with previously determined gene sequences, establish the complete protein sequences for Arc (53 residues) and Mnt (82 residues). Both proteins have melting temperatures between 45 and 55 degrees C and can be renatured to a fully active species. Arc is a dimer in solution and Mnt is a tetramer.

Amino Acid Sequence↗

PubMed1985

Crystallization of the Arc repressor.

Arc, a repressor from Salmonella phage P22 has been crystallized from ammonium phosphate at pH 8.0. The crystals form in space group P212121 with a = 90.26 A, b = 52.88 A and c = 47.58 A. The crystals diffract to 2.2 A resolution.

Crystallography↗