Biology subjects

Eustermann, S.

Publications and source records attributed to Eustermann, S..

8 recordsLinked to original sources

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↗

bioRxiv2024

EasyGrid: A versatile platform for automated cryo-EM sample preparation and quality control

Imaging biological macromolecules in their native state with single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography (cryo-ET) requires optimized approaches for the preparation and vitrification of biological samples. Here, we describe EasyGrid, a versatile technology enabling systematic, tailored and advanced sample preparation for cellular and structural biology. This automated, standalone platform combines in-line plasma treatment, microfluidic dispensing, blot-less sample spreading, jet-based vitrification and on-the-fly grid quality control using light interferometry to streamline cryo-EM sample optimization. With EasyGrid, we optimized grid preparation for different purified macromolecular complexes and subsequently determined their structure with cryo-EM. We also demonstrated how the platform allows better vitrification of large, mammalian cells compared to standard plunge-freezing. Automated sample preparation with EasyGrid establishes an advanced, high-throughput platform for both single-particle cryo-EM and cellular cryo-ET sample preparation.

molecular biology↗

bioRxiv2023

Structural insights into human TFIIIC promoter recognition

Transcription factor IIIC (TFIIIC) recruits RNA polymerase (Pol) III to most of its target genes. Recognition of intragenic A- and B-box motifs in tRNA genes by TFIIIC modules {tau}A and {tau}B is the first critical step for tRNA synthesis but is mechanistically poorly understood. Here, we report cryo-EM structures of the human 624 kDa TFIIIC complex unbound and bound to a tRNA gene. The {tau}B module recognizes the B-box via DNA shape and sequence readout through the assembly of multiple winged-helix domains. TFIIIC220 forms an integral part of both {tau}A and {tau}B connecting the two subcomplexes via a [~]550 amino acid residue flexible linker. Our data provide a structural mechanism by which high-affinity B-box recognition anchors TFIIIC to promoter DNA and permits scanning for low-affinity A-boxes and TFIIIB for Pol III activation.

molecular biology↗

bioRxiv2022

Structural dynamics of DNA strand break sensing by PARP-1 at a single-molecule level

Single stranded breaks (SSBs) are the most frequent DNA lesions threatening genomic integrity. A highly kinked DNA structure in complex with human PARP-1 led to the proposal that SSB sensing in Eukaryotes relies on dynamics of both the broken DNA double helix and PARP-1s multi-domain organization. Here, we directly probe this fundamental yet poorly understood process at the single-molecule level. Quantitative SM-FRET and structural ensemble calculations reveal how PARP-1 binding converts DNA SSBs from a largely unperturbed conformation into the highly kinked state. Binding of the second N-terminal zinc finger yields an intermediate DNA conformation that can be recognized by the first zinc finger. Thus, the data is inconsistent with a conformational selection model, instead an induced fit mechanism via a multi-domain assembly cascade drives SSBs sensing. Interestingly, a clinically used PARP-1 inhibitor niraparib shifts the equilibrium towards the unkinked state, whereas the inhibitor EB47 stabilizes the kinked state.

biophysics↗

bioRxiv2021

A hold-and-feed mechanism drives directional DNA loop extrusion by condensin

SMC protein complexes structure genomes by extruding DNA loops, but the molecular mechanism that underlies their activity has remained unknown. We show that the active condensin complex entraps the bases of a DNA loop in two separate chambers. Single-molecule and cryo-electron microscopy provide evidence for a power-stroke movement at the first chamber that feeds DNA into the SMC-kleisin ring upon ATP binding, while the second chamber holds on upstream of the same DNA double helix. Unlocking the strict separation of motor and anchor chambers turns condensin from a one-sided into a bidirectional DNA loop extruder. We conclude that the orientation of two topologically bound DNA segments during the course of the SMC reaction cycle determines the directionality of DNA loop extrusion.

biochemistry↗

bioRxiv2020

Genome information processing by the INO80 chromatin remodeler positions nucleosomes

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin.

biochemistry↗

bioRxiv2020

Measuring DNA mechanics on the genome scale

Mechanical deformations of DNA such as bending are ubiquitous and implicated in diverse cellular functions1. However, the lack of high-throughput tools to directly measure the mechanical properties of DNA limits our understanding of whether and how DNA sequences modulate DNA mechanics and associated chromatin transactions genome-wide. We developed an assay called loop-seq to measure the intrinsic cyclizability of DNA - a proxy for DNA bendability - in high throughput. We measured the intrinsic cyclizabilities of 270,806 50 bp DNA fragments that span the entire length of S. cerevisiae chromosome V and other genomic regions, and also include random sequences. We discovered sequence-encoded regions of unusually low bendability upstream of Transcription Start Sites (TSSs). These regions disfavor the sharp DNA bending required for nucleosome formation and are co-centric with known Nucleosome Depleted Regions (NDRs). We show biochemically that low bendability of linker DNA located about 40 bp away from a nucleosome edge inhibits nucleosome sliding into the linker by the chromatin remodeler INO80. The observation explains how INO80 can create promoter-proximal nucleosomal arrays in the absence of any other factors2 by reading the DNA mechanical landscape. We show that chromosome wide, nucleosomes are characterized by high DNA bendability near dyads and low bendability near the linkers. This contrast increases for nucleosomes deeper into gene bodies, suggesting that DNA mechanics plays a previously unappreciated role in organizing nucleosomes far from the TSS, where nucleosome remodelers predominate. Importantly, random substitution of synonymous codons does not preserve this contrast, suggesting that the evolution of codon choice has been impacted by selective pressure to preserve sequence-encoded mechanical modulations along genes. We also provide evidence that transcription through the TSS-proximal nucleosomes is impacted by local DNA mechanics. Overall, this first genome-scale map of DNA mechanics hints at a mechanical code with broad functional implications.

biophysics↗

bioRxiv2020

Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the ruler that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

biochemistry↗