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1.
Nucleic Acids Res ; 52(5): 2260-2272, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38109289

RESUMO

Intrinsically disordered regions (IDRs) are abundant in eukaryotic proteins, but their sequence-function relationship remains poorly understood. IDRs of transcription factors (TFs) can direct promoter selection and recruit coactivators, as shown for the budding yeast TF Msn2. To examine how IDRs encode both these functions, we compared genomic binding specificity, coactivator recruitment, and gene induction amongst a large set of designed Msn2-IDR mutants. We find that both functions depend on multiple regions across the > 600AA IDR. Yet, transcription activity was readily disrupted by mutations that showed no effect on the Msn2 binding specificity. Our data attribute this differential sensitivity to the integration of a relaxed, composition-based code directing binding specificity with a more stringent, motif-based code controlling the recruitment of coactivators and transcription activity. Therefore, Msn2 utilizes interwoven sequence grammars for encoding multiple functions, suggesting a new IDR design paradigm of potentially general use.


Assuntos
Proteínas de Ligação a DNA , Proteínas Intrinsicamente Desordenadas , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Regulação da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Mutação , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo
2.
Nucleic Acids Res ; 2024 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-38908024

RESUMO

Intrinsically disordered regions (IDRs) guide transcription factors (TFs) to their genomic binding sites, raising the question of how structure-lacking regions encode for complex binding patterns. We investigated this using the TF Gln3, revealing sets of IDR-embedded determinants that direct Gln3 binding to respective groups of functionally related promoters, and enable tuning binding preferences between environmental conditions, phospho-mimicking mutations, and orthologs. Through targeted mutations, we defined the role of short linear motifs (SLiMs) and co-binding TFs (Hap2) in stabilizing Gln3 at respiration-chain promoters, while providing evidence that Gln3 binding at nitrogen-associated promoters is encoded by the IDR amino-acid composition, independent of SLiMs or co-binding TFs. Therefore, despite their apparent simplicity, TF IDRs can direct and regulate complex genomic binding patterns through a combination of SLiM-mediated and composition-encoded interactions.

3.
Nucleic Acids Res ; 52(10): 5720-5731, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38597680

RESUMO

The Origin Recognition Complex (ORC) seeds replication-fork formation by binding to DNA replication origins, which in budding yeast contain a 17bp DNA motif. High resolution structure of the ORC-DNA complex revealed two base-interacting elements: a disordered basic patch (Orc1-BP4) and an insertion helix (Orc4-IH). To define the ORC elements guiding its DNA binding in vivo, we mapped genomic locations of 38 designed ORC mutants, revealing that different ORC elements guide binding at different sites. At silencing-associated sites lacking the motif, ORC binding and activity were fully explained by a BAH domain. Within replication origins, we reveal two dominating motif variants showing differential binding modes and symmetry: a non-repetitive motif whose binding requires Orc1-BP4 and Orc4-IH, and a repetitive one where another basic patch, Orc1-BP3, can replace Orc4-IH. Disordered basic patches are therefore key for ORC-motif binding in vivo, and we discuss how these conserved, minor-groove interacting elements can guide specific ORC-DNA recognition.


Assuntos
Complexo de Reconhecimento de Origem , Origem de Replicação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sítios de Ligação , Replicação do DNA , DNA Fúngico/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , Mutação , Motivos de Nucleotídeos , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/química , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química
4.
Methods Mol Biol ; 2846: 263-283, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39141241

RESUMO

Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles.


Assuntos
Genoma Fúngico , Sequenciamento de Nucleotídeos em Larga Escala , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Cromatina/genética , Cromatina/metabolismo , Sítios de Ligação , Análise de Sequência de DNA/métodos , Nuclease do Micrococo/metabolismo , Nuclease do Micrococo/genética , Biologia Computacional/métodos
5.
bioRxiv ; 2024 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-38766039

RESUMO

Contact-sites are specialized zones of proximity between two organelles, essential for organelle communication and coordination. The formation of contacts between the Endoplasmic Reticulum (ER), and other organelles, relies on a unique membrane environment enriched in sterols. However, how these sterol-rich domains are formed and maintained had not been understood. We found that the yeast membrane protein Yet3, the homolog of human BAP31, is localized to multiple ER contact sites. We show that Yet3 interacts with all the enzymes of the post-squalene ergosterol biosynthesis pathway and recruits them to create sterol-rich domains. Increasing sterol levels at ER contacts causes its depletion from the plasma membrane leading to a compensatory reaction and altered cell metabolism. Our data shows that Yet3 provides on-demand sterols at contacts thus shaping organellar structure and function. A molecular understanding of this protein's functions gives new insights into the role of BAP31 in development and pathology.

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