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Biomedical subjects

A Schepartz

Publications and source records attributed to A Schepartz.

30 records · Page 2Linked to original sources
PubMed1997

Inhibition of Rev·RRE complexation by triplex tethered oligonucleotide probes.

We have described a class of molecules, called tethered oligonucleotide probes (TOPs), that bind RNA on the basis of both sequence and structure. TOPs consist of two short oligonucleotides joined by a tether whose length and composition may be varied using chemical synthesis. In a triplex TOP, one oligonucleotide recognizes a short single-stranded region in a target RNA through the formation of Watson-Crick base pairs; the other oligonucleotide recognizes a short double-stranded region through the formation of Hoogsteen base pairs. Binding of triplex TOPs to an HIV-1 Rev Response Element RNA variant (RREAU) was measured by competition electrophoretic mobility shift analysis. Triplex TOP.RREAU stabilities ranged between -9.6 and -6.1 kcal mol-1 under physiological conditions of pH, salt, and temperature. Although the most stable triplex TOP.RREAU complex contained 12 contiguous U.AU triple helical base pairs, complexes containing only six or nine triple helical base pairs also formed. Triplex TOPs inhibited formation of the RRE.Rev complex with IC50 values that paralleled the dissociation constants of the analogous triplex TOP.RREAU complexes. In contrast to results obtained with TOPs that target two single-stranded RRE regions, inhibition of Rev.RREAU complexation by triplex TOPs did not require pre-incubation of RREAU and a TOP: triplex TOPs competed efficiently with Rev for RREAU and inhibited RREAU.Rev complexation at equilibrium.

Base Composition↗
PubMed1995

Mechanism of DNA-binding enhancement by the human T-cell leukaemia virus transactivator Tax.

Tax protein activates transcription of the human T-cell leukaemia virus type I (HTLV-I) genome through three imperfect cyclic AMP-responsive element (CRE) target sites located within the viral promoter. Previous work has shown that Tax interacts with the bZIP element of proteins that bind the CRE target site to promote peptide dimerization, suggesting an association between Tax and bZIP coiled coil. Here we show that the site of interaction with Tax is not the coiled coil, but the basic segment. This interaction increases the stability of the GCN4 bZIP dimer by 1.7 kcal mol-1 and the DNA affinity of the dimer by 1.9 kcal mol-1. The differential effect of Tax on several bZip-DNA complexes that differ in peptide sequence or DNA conformation suggests a model for Tax action based on stabilization of a distinct DNA-bound protein structure. This model may explain how Tax interacts with transcription factors of considerable sequence diversity to alter patterns of gene expression.

Amino Acid Sequence↗
PubMed1995

Conformation of Tax-response elements in the human T-cell leukemia virus type I promoter.

BACKGROUND: HTLV-I Tax is believed to activate viral gene expression by binding bZIP proteins (such as CREB) and increasing their affinities for proviral TRE target sites. Each 21 bp TRE target site contains an imperfect copy of the intrinsically bent CRE target site (the TRE core) surrounded by highly conserved flanking sequences. These flanking sequences are essential for maximal increases in DNA affinity and transactivation, but they are not, apparently, contacted by protein. Here we employ non-denaturing gel electrophoresis to evaluate TRE conformation in the presence and absence of bZIP proteins, and to explore the role of DNA conformation in viral transactivation. RESULTS: Our results show that the TRE-1 flanking sequences modulate the structure and modestly increase the affinity of a CREB bZIP peptide for the TRE-1 core recognition sequence. These flanking sequences are also essential for a maximal increase in stability of the CREB-DNA complex in the presence of Tax. CONCLUSIONS: The CRE-like TRE core and the TRE flanking sequences are both essential for formation of stable CREB-TRE-1 and Tax-CREB-TRE-1 complexes. These two DNA segments may have co-evolved into a unique structure capable of recognizing Tax and a bZIP protein.

Activating Transcription Factor 1↗
PubMed1994

Mapping RNA regions in eukaryotic ribosomes that are accessible to methidiumpropyl-EDTA.Fe(II) and EDTA.Fe(II).

Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and EDTA.Fe(II) were used to investigate the structure of Drosophila melanogaster ribosomes. Cleavage reactions were performed on intact ribosomes in cell lysates in vitro and analyzed by primer extension with reverse transcriptase using oligodeoxynucleotide primers. Regions of 18S and 28S ribosomal RNAs (rRNAs) which are accessible to MPE.Fe(II) and EDTA.Fe(II) are located almost exclusively within expansion segments. The accessibility of these regions to cleavage indicates that they are likely exposed on the surface of eukaryotic ribosomes. These results provide information about the overall tertiary structure of rRNA in ribosomes.

Animals↗
PubMed1994

DNA targets for certain bZIP proteins distinguished by an intrinsic bend.

In spite of the large amount of sequence conservation among the DNA binding segments of basic region leucine zipper (bZIP) proteins, these proteins can discriminate differently between target sequences that differ in half-site spacing. Here it is shown that the half-site spacing preferences of bZIP proteins are the result of (i) the differential intrinsic curvature in target binding sites that differ by insertion or deletion of a single base pair and (ii) the ability of some bZIP proteins to overcome this intrinsic curvature through a mechanism dependent on basic segment residues.

Activating Transcription Factor 2↗
PubMed1994

Distribution of labor among bZIP segments in the control of DNA affinity and specificity.

BACKGROUND: The basic region-leucine zipper (bZIP) family of proteins use an atypically simple motif for DNA recognition, yet family members discriminate differently between target sites that differ only in half-site spacing. Two such sites are the cAMP-response element (CRE) and the AP-1 target site. Fos/Jun prefers the AP-1 site (ATGACTCAT), while CRE-BP1 prefers CRE (ATGACGTCAT), and GCN4 binds both sites with equal affinity. We therefore asked what determines the relative specificity for CRE and AP-1 sites in bZIP proteins. RESULTS: Here we show that CRE/AP-1 specificity in CRE-BP1 is encoded within the spacer and basic segments of the bZIP element. Of these two regions, the basic segment is the more important. This specificity is in part achieved at the expense of affinity. CONCLUSIONS: The small size and simplicity of the bZIP recognition helix was already unusual; our findings show that the information that determines the target site specificity of members of the bZIP family of proteins is even more condensed than expected. These results suggest that it may be possible to design surprisingly small proteins that bind DNA with high sequence specificity, although it may be more difficult to achieve high-affinity binding in small proteins.

Amino Acid Sequence↗
PubMed1993

Design of a metallo-bZIP protein that discriminates between CRE and AP1 target sites: selection against AP1.

The bZIP class of dimeric DNA binding proteins is characterized by a motif containing two discrete domains: a DNA contact domain defined by conserved basic and hydrophobic residues (basic domain), and a dimerization domain identified by a heptad repeat of leucine residues (zipper domain). Molecules are constructed in which the GCN4 dimerization domain is replaced by a series of stereochemically defined metal ion complexes that alter systematically the relative orientation of the basic domain peptides. Both the affinity and the specificity of DNA binding are modulated by seemingly small changes in metal complex stereochemistry. Although GCN4 binds CRE (ATGACGTCAT) and AP1 (ATGACTCAT) target sites with comparable affinity, one metallo-bZIP peptide ([G29Ts]2Fe) prefers the CRE by 4 kcal.mol-1 (1 cal = 4.184 J). Competition experiments performed with several DNAs demonstrate that discrimination between CRE and AP1 is dominated by selection against the AP1 site.

Amino Acid Sequence↗
PubMed1993

Altered specificity of DNA-binding proteins with transition metal dimerization domains.

The bZIP motif is characterized by a leucine zipper domain that mediates dimerization and a basic domain that contacts DNA. A series of transition metal dimerization domains were used to alter systematically the relative orientation of basic domain peptides. Both the affinity and the specificity of the peptide-DNA interaction depend on domain orientation. These results indicate that the precise configuration linking the domains is important; dimerization is not always sufficient for DNA binding. This approach to studying the effect of orientation on protein function complements mutagenesis and could be used in many systems.

Amino Acid Sequence↗
PubMed1992

Conformation-dependent cleavage of staphylococcal nuclease with a disulfide-linked iron chelate.

We report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide. By using this easily prepared cysteine-specific hydrophilic reagent, an ethylenediaminetriacetic acid-Fe3+ complex (EDTA-Fe) was covalently attached to a single genetically engineered cysteine residue in staphylococcal nuclease. Upon addition of the iron reductant ascorbate, the nuclease-EDTA-Fe conjugate underwent a protein self-cleavage reaction mediated by reactive oxygen species. Sequence analysis of the products indicated that cleavage occurs close in tertiary structure to the EDTA-Fe attachment site. In the presence of denaturants, the cleavage pattern changes and the reaction is limited to residues proximal in sequence to the cysteine attachment site. These results indicate that intramolecular protein cleavage reactions mediated by EDTA-Fe can be used to evaluate changes in protein conformation. The reagent described should be a useful tool in the structural mapping of nonnative protein states populated at equilibrium, such as the molten globule, that are frequently refractory to conventional structure analysis.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Gene regulation: protein escorts to the transcription ball.

A new way by which the potency of a eukaryotic transcription factor can be regulated has been discovered, in which nuclear factors increase the concentration of the transcription factor's active form by modulating an otherwise unfavorable equilibrium between monomeric and dimeric forms of the protein.

DNA↗