Biology subjects

Müller, C. W.

Publications and source records attributed to Müller, C. W..

7 recordsLinked to original sources

bioRxiv2024

A photoswitchable HaloTag for spatiotemporal control of fluorescence in living cells

Photosensitive fluorophores, which emission can be controlled using light, are essential for advanced biological imaging, enabling precise spatiotemporal tracking of molecular features, and facilitating super-resolution microscopy techniques. While irreversibly photoactivatable fluorophores are well established, reversible reporters which can be re-activated multiple times remain scarce, and only few have been applied in living cells using generalizable protein labelling methods. To address these limitations, we introduce chemigenetic photoswitchable fluorophores, leveraging the self-labelling HaloTag protein with fluorogenic rhodamine dye ligands. By incorporating a light-responsive protein domain into HaloTag, we engineer a tunable, photoswitchable HaloTag (psHaloTag), which can reversibly modulate the fluorescence of a bound dye-ligand via a light-induced conformational change. Our best performing psHaloTag variants show high performance in vitro and in living cells, with large, reversible, far-red fluorescence turn-on upon 450 nm illumination across various biomolecular targets. Together, this work establishes the chemigenetic approach as a versatile platform for the design of photoswitchable reporters, tunable through both genetic and synthetic modifications, with promising applications for dynamic imaging.

biochemistry↗

bioRxiv2022

Mechanism of RNA Polymerase I selection by transcription factor UAF

Pre-ribosomal RNA is selectively transcribed by RNA Polymerase (Pol) I in eukaryotes. The yeast transcription factor Upstream Activating Factor (UAF) represses Pol II transcription and mediates Pol I preinitiation complex (PIC) formation during the early stages of transcription initiation at the 35S ribosomal RNA gene. To unravel the DNA recognition and Pol I selection mechanisms of UAF, we determined the structure of UAF bound to native promoter DNA and transcription factor TBP. We found that UAF recognizes DNA using a hexameric histone-like scaffold with markedly different interactions than the nucleosome and the histone-fold-rich TFIID. UAF strategically sequesters TBP from DNA and Pol II/III-specific factors, and positions it for Core Factor binding, supporting Pol I recruitment. Our findings therefore reveal the molecular basis of Pol I selection for ribosome biogenesis. As well, they reveal an unexpected potential within the histone fold as a motif for specific protein-DNA interactions inside the cell.

molecular biology↗

bioRxiv2021

The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

Numerous chromatin-associated proteins have been linked to neurodevelopmental disorders, yet their molecular functions often remain elusive. PHF14, HMG20A, TCF20 and RAI1 are components of a putative chromatin-associated complex and have been implicated in neurological disorders. Here, we found that Phf14 knockout embryonic stem cells and neural progenitor cells exhibit impaired cell cycle progression and proliferation, inadequate protection of stalled replication forks, and decreased DNA repair. The PHF14 complex rapidly assembles at DNA damage sites and binds to DNA through HMG20A. The PHF14 complex forms DNA-containing phase separated droplets in vitro, where TCF20 facilitates droplet formation. Furthermore, TCF20 maintenance at DNA damage sites is destabilized upon pathological mutation. Our results suggest that the PHF14 complex contributes to DNA damage repair by sensing damaged sites and forming biomolecular condensates, thus supporting cell cycle progression, especially in neural progenitor cells whose spatiotemporal pool is critical for proper brain development.

molecular biology↗

bioRxiv2021

Cryo-EM structures of human RNA polymerase I

RNA polymerase I (Pol I) specifically synthesizes ribosomal RNA. Pol I upregulation is linked to cancer, while mutations in the Pol I machinery lead to developmental disorders. Here, we report the cryo-EM structure of elongating human Pol I at 2.7 [A] resolution. In the exit tunnel, we observe a double-stranded RNA helix that may support Pol I processivity. Our structure confirms that human Pol I consists of 13 subunits with only one subunit forming the Pol I stalk. Additionally, the structure of human Pol I in complex with the initiation factor RRN3 at 3.1 [A] resolution reveals stalk flipping upon RRN3 binding. We also observe an inactivated state of human Pol I bound to an open DNA scaffold at 3.3 [A] resolution. Lastly, the high-resolution structure of human Pol I allows mapping of disease-related mutations that can aid understanding of disease etiology.

molecular biology↗

bioRxiv2020

Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states

ABSTRACTRNA polymerase III (Pol III) synthesises tRNAs and other short, essential RNAs. Human Pol III misregulation is linked to tumour transformation, neurodegenerative and developmental disorders, and increased sensitivity to viral infections. Pol III inhibition increases longevity in different animals but also promotes intracellular bacterial growth owing to its role in the immune system. This highlights the importance to better understand human Pol III transcription on a molecular level. Here, we present cryo-EM structures at 2.8 to 3.3 Å resolution of transcribing and unbound human Pol III purified from human suspension cells that were gene-edited by CRISPR-Cas9. We observe insertion of the TFIIS-like subunit RPC10 into the polymerase funnel, providing insights into how RPC10 triggers transcription termination. Our structures also resolve elements absent from S. cerevisiae Pol III such as the winged-helix domains of RPC5 and an iron-sulphur cluster in RPC6, which tethers the heterotrimer subcomplex to the Pol III core. The cancer-associated RPC7α isoform binds the polymerase clamp, potentially interfering with Pol III inhibition by the tumour suppressor MAF1, which may explain why overexpressed RPC7α enhances tumour transformation. Finally, the human Pol III structure allows mapping of disease-related mutations and might contribute to developing inhibitors that selectively target Pol III for therapeutic interventions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

Structure of the TFIIIC subcomplex {tau} A provides insights into RNA polymerase III pre-initiation complex formation

Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex {tau}A, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that {tau}A binding to its promoter is auto-inhibited by a disordered acidic tail of subunit {tau}95. We further provide a negative stain reconstruction of {tau}A bound to the TFIIIB subunits Brf1 and TBP with an unexpected location of Brf1 and TBP. This shows that a ruler element in {tau}A achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.

biochemistry↗

bioRxiv2019

Structural basis of RNA Polymerase III transcription repression by Maf1

Maf1 is a highly conserved central regulator of transcription by RNA polymerase III (Pol III), and Maf1 activity influences a wide range of phenotypes from metabolic efficiency to lifespan. Here, we present a 3.3 [A] cryo-EM structure of yeast Maf1 bound to Pol III, which establishes how Maf1 achieves transcription repression. In the Maf1-bound state, Pol III elements that are involved in transcription initiation are sequestered, and the active site is sealed off due to ordering of the mobile C34 winged helix 2 domain. Specifically, the Maf1 binding site overlaps with the binding site of the Pol III transcription factor TFIIIB and DNA in the pre-initiation complex, rationalizing that binding of Maf1 and TFIIIB to Pol III are mutually exclusive. We validate our structure using variants of Maf1 with impaired transcription-inhibition activity. These results reveal the exact mechanism of Pol III inhibition by Maf1, and rationalize previous biochemical data.

molecular biology↗