Biology subjects

Baudin, F.

Publications and source records attributed to Baudin, F..

9 recordsLinked to original sources

bioRxiv2025

Recruitment of Fpt1 to tRNA genes requires TFIIIB and the N-terminal TPR array of TFIIIC subunit {tau}131

Transfer-RNA genes (tDNAs) in budding yeast recruit varying amounts of Fpt1, a regulator of RNA polymerase III (RNAPIII) occupancy. Fpt1 occupancy resembles that of the general transcription factor TFIIIC but how Fpt1 is recruited to tDNAs remains unclear. Here we show that both TFIIIB and TFIIIC are required for Fpt1 binding under active as well as repressive RNAPIII conditions. Depletion of TFIIIB reduced Fpt1 occupancy without affecting TFIIIC. In contrast, TFIIIC depletion led to reduced Fpt1 and a gene-specific reduction in TFIIIB occupancy. Moreover, upon depletion of TFIIIC, Fpt1 and TFIIIB were lost to different extents. We identified the C-terminal intrinsically disordered region of Fpt1 as critical for increased Fpt1 binding under repressive conditions. Within this region, a short -helix was predicted to interact with the N-terminal tetratricopeptide repeat array of the TFIIIC subunit {tau}131, a region also known to interact with TFIIIB. Deletion of this -helix abrogated stress-induced Fpt1 recruitment to tDNAs, as did mutations in the predicted interaction surface of {tau}131, while having a milder effect on TFIIIB occupancy. Together, these findings uncovered a dual and dynamic mechanism of Fpt1 recruitment to tDNAs with independent contributions of TFIIIB and TFIIIC.

molecular biology↗

bioRxiv2024

Structural and kinetic insights into tRNA promoter engagement by yeast general transcription factor TFIIIC

Transcription of tRNA genes by RNA polymerase III requires the general transcription factor IIIC (TFIIIC), which recognizes intragenic A-box and B-box DNA motifs of type II gene promoters. However, the underlying mechanism has remained elusive, in part due to missing structural information for A-box recognition. In this study, we use single-particle cryo-EM and single-molecule FRET (smFRET) to reveal structural and real-time kinetic insights into how the 520 kDa yeast TFIIIC complex engages A- and B-box DNA motifs in the context of a tRNA gene promoter. Cryo-EM structures of {tau}A and {tau}B subcomplexes bound to the A- and B-box were obtained at 3.7 and 2.5 [A] resolution, respectively, while cryo-EM single particle mapping determined the specific distance and relative orientation of the {tau}A and {tau}B subcomplexes revealing a fully engaged state of TFIIIC. smFRET experiments show that overall recruitment and residence times of TFIIIC on a tRNA gene are primarily governed by B-box recognition, while footprinting experiments suggest a key role of {tau}A and the A-box in TFIIIB and Pol III recruitment following TFIIIC recognition of type II promoters.

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

Structural snapshots of La Crosse virus polymerase reveal the mechanisms underlying Peribunyaviridae replication and transcription

Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse virus polymerase and visualize by cryo-electron microscopy its conformational dynamics, unveiling the precise molecular mechanics underlying its essential activities. Replication initiation is coupled to distal duplex promoter formation, endonuclease movement, prime-and-realign loop extension and closure of the polymerase core that direct the template towards the active site. Transcription initiation depends on C-terminal region closure and endonuclease movements that firstly prompt primer cleavage and secondly promote primer entry in the active site. Product realignment after priming, observed in replication and transcription, is triggered by the prime-and-realign loop. Switch to elongation results in polymerase reorganization and core region opening to facilitate template-product duplex formation in the active site cavity. The detailed uncovered mechanics will be crucial for future design of antivirals counteracting bunyaviral life-threatening pathogens.

microbiology↗

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↗

bioRxiv2021

Mechanism of riboregulation of p62 protein oligomerisation by vault RNA1-1 in selective autophagy

Macroautophagy ensures the clearance of intracellular substrates ranging from single ubiquitinated proteins to large proteotoxic aggregates and defective organelles. The selective autophagy receptor p62 binds these targets and recruits them to double-membrane vesicles, which fuse with lysosomes to degrade their content. We recently uncovered that p62 function is riboregulated by the small non-coding vault RNA1-1. Here, we present detailed insight into the underlying mechanism. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within the RNA that mediates the specific p62 interaction. Our data define molecular determinants that govern mammalian autophagy via the p62-vault RNA1-1 riboregulatory pair.

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↗