Search PubMed⌕ Search

Biomedical subjects

Aseem Z Ansari

Publications and source records attributed to Aseem Z Ansari.

12 recordsLinked to original sources
PubMed2005

Unraveling the mechanism of a potent transcriptional activator.

Despite their enormous potential as novel research tools and therapeutic agents, artificial transcription factors (ATFs) that up-regulate transcription robustly in vivo remain elusive. In investigating an ATF that does function exceptionally well in vivo, we uncovered an unexpected relationship between transcription function and a binding interaction between the activation domain and an adjacent region of the DNA binding domain. Disruption of this interaction leads to complete loss of function in vivo, even though the activation domain is still able to bind to its target in the transcriptional machinery. We propose that this interaction parallels those between natural activation domains and their regulatory proteins, concealing the activation domain from solvent and the cellular milieu until it binds to its transcriptional machinery target. Inclusion of this property in the future design of ATFs should enhance their efficacy in vivo.

Amino Acid Sequence↗
PubMed2005

Temperature-sensitive protein-DNA dimerizers.

Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription factors. A hairpin polyamide-YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. Such small molecules function as protein-DNA dimerizers that stabilize complexes at composite DNA binding sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial degree of variability in the linker length is tolerated at lower temperatures. At physiological temperatures, the longest linker tested confers a "switch"-like property on the protein-DNA dimerizer, in that it abolishes the ability of the YPWM moiety to recruit the natural transcription factor to DNA. These observations provide design principles for future artificial transcription factors that can be externally regulated and can function in concert with the cellular regulatory circuitry.

Amino Acid Sequence↗
PubMed2005

Transcriptional activating regions target attached substrates to a cyclin-dependent kinase.

The yeast cyclin-dependent kinase Srb10 phosphorylates various transcriptional activators as they activate transcription, and acidic transcriptional activating domains found on several activators directly bind Srb10. Here we show that the interaction between Srb10 (with its associated cyclin Srb11) and each of several different activating regions, in vitro, leads to the phosphorylation of peptide sequences attached to but outside of the activating regions themselves. In some cases, residues within the activating regions are also phosphorylated. The results define a mechanism by which a kinase is recruited to alternate substrates with diverse physiological consequences.

Amino Acid Sequence↗
PubMed2004

Two cyclin-dependent kinases promote RNA polymerase II transcription and formation of the scaffold complex.

Three cyclin-dependent kinases, CDK7, -8, and -9, are specifically involved in transcription by RNA polymerase II (Pol II) and target the Pol II C-terminal domain (CTD). The role of CDK7 and CDK8 kinase activity in transcription has been unclear, with CDK7 shown to have variable effects on transcription and CDK8 suggested to repress transcription and/or to target other gene-specific factors. Using a chemical genetics approach, the Saccharomyces cerevisiae homologs of these kinases, Kin28 and Srb10, were engineered to respond to a specific inhibitor and the inhibitor was used to test the role of these kinases in transcription in vivo and in vitro. In vitro, these kinases can both promote transcription, with up to 70% of transcription abolished when both kinases are inhibited together. Similarly, in vivo inhibition of both kinases together gives the strongest decrease in transcription, as measured by chromatin immunoprecipitation of Pol II. Kin28 and Srb10 also have overlapping roles in promoting ATP-dependent dissociation of the preinitiation complex (PIC) into the Scaffold complex. Using the engineered kinases and an ATP analog, specific kinase substrates within the PIC were identified. In addition to the previously known substrate, the Pol II CTD, it was found that Kin28 phosphorylates two subunits of Mediator and Srb10 targets two subunits of TFIID for phosphorylation.

Cell-Free System↗
PubMed2003

Toward artificial developmental regulators.

A polyamide-peptide conjugate is designed which recruits sequence specifically the developmental regulator Exd to a cognate DNA site. In particular, an eight-ring hairpin polyamide (Im-Im-Py(C3H6NHR)-Py-gamma-Im-Py-Py-Py-beta-Dp) with a heptapeptide (R = Ac-Phe-Tyr-Pro-Trp-Met-Lys-Gly-) attached on a central ring was shown to induce cooperative binding of the Drosophila Hox protein cofactor Exd with a Kd of 4.4 nM in vitro, an order of magnitude more efficient than the natural Hox protein partner Ubx. The conjugate joins two sequence specific domains, one for DNA and one for the protein. This small molecule thus serves as a cooperative protein-DNA dimerizer, which mimics the natural Hox family of developmental regulators.

Biomimetic Materials↗
PubMed2003

RNA sequences that work as transcriptional activating regions.

We describe a set of RNA molecules that work as transcriptional activators when tethered to DNA. These RNA activating regions were found amongst a randomized set of molecules bearing variants of a 10 nt loop attached to an RNA stem. The various RNA activating regions all bear an identical five- residue sequence with an interspersed sixth residue. The result shows that although all natural activating regions characterized thus far are peptidic, this function can be served by other kinds of moieties as well.

Base Sequence↗
PubMed2002

Design of artificial transcriptional activators with rigid poly-L-proline linkers.

Typical eukaryotic transcriptional activators are composed of distinct functional domains, including a DNA binding domain and an activating domain. Artificial transcription factors have been designed wherein the DNA binding domain is a minor groove DNA binding hairpin polyamide linked by a flexible tether to short activating peptides, typically 16-20 residues in size. In this study, the linker between the polyamide and the peptide was altered in an incremental fashion using rigid oligoproline "molecular rulers" in the 18-45 A length range. We find that there is an optimal linker length which separates the DNA and the activation region for transcription activation.

Amino Acid Sequence↗
PubMed2002

Transcriptional activating regions target a cyclin-dependent kinase.

Several yeast activators are phosphorylated by SRB10, a cyclin-dependent kinase associated with the transcriptional machinery. Sites of phosphorylation are found outside the activating region in each case, and the modification has different physiological consequences in different cases. We show here that certain acidic transcriptional activating regions contact SRB10 as assayed both in vivo and in vitro. The interaction evidently positions each activator, as it activates transcription, so that it gets phosphorylated by SRB10, and thus a common mechanism targets disparate substrates to the kinase.

Cyclin-Dependent Kinase 8↗
PubMed2002

A target essential for the activity of a nonacidic yeast transcriptional activator.

P201 is a short (eight-residue) nonacidic peptide that comprises a strong transcriptional activating region when tethered to DNA in yeast. Here we identify the mediator protein Gal11 as an essential target of P201. Deletion of Gal11, which modestly decreases activation elicited by the typical acidic yeast activator, abolishes activation by DNA-tethered P201. A point mutation in Gal11, which has no effect on other Gal11 functions, also greatly diminishes activation by DNA-tethered P201. P201 binds to a fragment of Gal11 in vivo and in vitro, and the interaction is diminished by mutations in either component that decrease activation in vivo. P201, unlike the typical yeast acidic activating region, does not work in mammalian cells, which is consistent with the notion that the unique target of P201 (Gal11) is absent from mammalian cells.

Fungal Proteins↗
PubMed2002

Modular design of artificial transcription factors.

Eukaryotic transcription factors are composed of interchangeable modules. This has led to the design of a wide variety of modular artificial transcription factors (ATFs) that can stimulate or inhibit the expression of targeted genes. The ability to regulate the expression of any targeted gene using a 'programmable' ATF offers a powerful tool for functional genomics and bears tremendous promise in developing the field of transcription-based therapeutics.

Biotechnology↗