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Biomedical subjects

A Schepartz

Publications and source records attributed to A Schepartz.

At least 19 recordsLinked to original sources
PubMed1999

DNA specificity enhanced by sequential binding of protein monomers.

Transcriptional activation often requires the rapid assembly of complexes between dimeric transcription factors and specific DNA sites. Here we show that members of the basic region leucine zipper and basic region helix-loop-helix zipper transcription factor families follow an assembly pathway in which two protein monomers bind DNA sequentially and form their dimerization interface while bound to DNA. Nonspecific protein or DNA competitors have little effect on the rate of assembly along this pathway, but slow a competing pathway in which preformed dimers bind DNA. The sequential monomer-binding pathway allows the protein to search for and locate a specific DNA site more quickly, resulting in greater specificity prior to equilibrium.

Animals↗
PubMed1998

Sequence determinants of the intrinsic bend in the cyclic AMP response element.

The cyclic AMP response element (CRE site, ATGACGTCAT) is the DNA target for transcription factors whose activities are regulated by cyclic AMP (1). Recently, we discovered that the CRE site is bent by 10-13 degrees toward the major groove (2). Little or no bend is detected in the related AP-1 site (ATGACTCAT), which differs from the CRE site by loss of a single, central, C.G base pair (2, 3). Here we describe experiments designed to identify which base pairs within the CRE site induce the bent structure in an attempt to understand the origins of the dramatically different conformations of the CRE and AP-1 sites. Our data indicate that the intrinsic CRE bend results from distortion within the TGA sequence found in each CRE half site (ATGAC). These two TGA sequences are located in phase with one another in the CRE sequence but are not (completely) in phase in the AP-1 sequence. This difference in phasing leads to the overall difference in bend as detected by gel (2) and cyclization methods (S. C. Hockings, J. D. Kahn, and D. M. Crothers, unpublished results; M. A. Fabian and A. Schepartz, unpublished results). Our results confirm earlier predictions of altered structure within TG steps, provide insight into the structural reorganizations induced in DNA by bZIP proteins, and lead to a revision of the relationship between the structures of the free and bZIP-bound forms of the CRE and AP-1 sites.

Base Composition↗
PubMed1998

Preinitation complex assembly: potentially a bumpy path.

During 1996 and 1997, several chemical issues that arise in the early stages of preinitiation complex (PIC) formation were resolved. Kinetics experiments indicated that both TBP dimerization and DNA bending influence the rate of TBP-TATA box assembly. Affinity cleavage experiments indicated that TBP lacks the specificity to nucleate assembly of a properly oriented PIC. Finally, high-resolution structures provided the atomic detail of early intermediates in PIC formation.

DNA↗
PubMed1997

Mechanism of DNA binding enhancement by hepatitis B virus protein pX.

At least three hundred million people worldwide are infected with the hepatitis B virus (HBV), and epidemiological studies show a clear correlation between chronic HBV infection and the development of hepatocellular carcinoma. HBV encodes a protein, pX, which abducts the cellular transcriptional machinery in several ways including direct interactions with bZIP transcription factors. These interactions increase the DNA affinities of target bZIP proteins in a DNA sequence-dependent manner. Here we use a series of bZIP peptide models to explore the mechanism by which pX interacts with bZIP proteins. Our results suggest that pX increases bZIP.DNA stability by increasing the stability of the bZIP dimer as well as the affinity of the dimer for DNA. Additional experiments provide evidence for a mechanism in which pX recognizes the composite structure of the peptide.DNA complex, not simply the primary peptide sequence. These experiments provide a framework for understanding how pX alters the patterns of transcription within the nucleus. The similarities between the mechanism proposed for pX and the mechanism previously proposed for the human T-cell leukemia virus protein Tax are discussed.

Amino Acid Sequence↗
PubMed1997

Bidirectional binding of the TATA box binding protein to the TATA box.

By selective attachment of a DNA cleavage agent to specific residues in the yeast TATA box binding protein (yTBP), we demonstrate that, in solution, yTBP binds to the TATA boxes of both the adenovirus major late promoter and the yeast CYC1 promoter with only a modest preference in orientation and binds well to several overlapping binding sites. The general factors TFIIA and TFIIB each increase the rotational and translational selectivity of yTBP but are not sufficient, at least individually, to confer a unique polarity to the preinitiation complex. We conclude that TBP alone cannot define the productive orientation of general factor assembly on a promoter.

Adenoviridae↗
PubMed1997

Electrostatic mechanism for DNA bending by bZIP proteins.

Biology is replete with examples of protein-induced DNA bending, yet the forces responsible for bending have been neither established nor quantified. Mirzabekov and Rich proposed in 1979 that asymmetric neutralization of the anionic phosphodiester backbone by basic histone proteins could provide a thermodynamic driving force for DNA bending in the nucleosome core particle [Mirzabekov, A. D., & Rich, A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1118-1121]. Strauss and Maher lent support to this proposal in 1994 by demonstrating that replacement of six proximal phosphate residues with neutral methylphosphonates resulted in DNA bent spontaneously toward the neutralized face [Strauss, J. K., & Maher, L. J., III (1994) Science 266, 1829-1834; Strauss, J. K., Prakash, T. P., Roberts, C., Switzer, C., & Maher, L. J., III (1996) Chem. Biol. 3, 671-678; Strauss, J. K., Roberts, C.; Nelson, M. G.; Switzer, C., & Maher, J. L., III (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9515-9520]. Here it is shown that bZIP proteins bend DNA via a mechanism involving direct contacts between one or two basic side chains and a symmetry-related pair of unique, nonbridging phosphate oxygens. The locations of these phosphates provide direct experimental support for a protein-induced bending mechanism based on asymmetric charge neutralization. This straightforward mechanism is compatible with many DNA-recognition motifs and may represent a general strategy for the assembly of protein-DNA complexes of defined stereochemistries.

Amino Acid Sequence↗
PubMed1997

The role of a basic amino acid cluster in target site selection and non-specific binding of bZIP peptides to DNA.

The ability of a transcription factor to locate and bind its cognate DNA site in the presence of closely related sites and a vast array of non-specific DNA is crucial for cell survival. The CREB/ATF family of transcription factors is an important group of basic region leucine zipper (bZIP) proteins that display high affinity for the CRE site and low affinity for the closely related AP-1 site. Members of the CREB/ATF family share in common a cluster of basic amino acids at the N-terminus of their bZIP element. This basic cluster is necessary and sufficient to cause the CRE site to bend upon binding of a CREB/ATF protein. The possibility that DNA bending and CRE/AP-1 specificity were linked in CREB/ATF proteins was investigated using chimeric peptides derived from human CRE-BP1 (a member of the CREB/ATF family) and yeast GCN4, which lacks both a basic cluster and CRE/AP-1 specificity. Gain of function and loss of function experiments demonstrated that the basic cluster was not responsible for the CRE/AP-1 specificity displayed by all characterized CREB/ATF proteins. The basic cluster was, however, responsible for inducing very high affinity for non- specific DNA. It was further shown that basic cluster-containing peptides bind non-specific DNA in a random coil conformation. We postulate that the high non- specific DNA affinities of basic cluster-containing peptides result from cooperative electrostatic interactions with the phosphate backbone that do not require peptide organization.

Activating Transcription Factor 2↗
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↗