Biology subjects

Hausner, W.

Publications and source records attributed to Hausner, W..

4 recordsLinked to original sources

bioRxiv2026

Structural basis of nucleosome remodeling by archaeal RNA polymerase during transcription elongation

Transcription occurs in the context of histone-based chromatin in eukaryotes and most archaea. The archaeal RNA polymerase and histones are ancestral to their eukaryotic counterparts, yet how RNA polymerase traverses histone-bound DNA in archaea remains poorly understood. Here, we reconstitute a nucleosome-associated transcription elongation complex (TEC) from Pyrococcus furiosus and capture its structure across multiple elongation states by cryo-electron microscopy (cryo-EM). High-resolution structures reveal that the RNA polymerase engages a three-dimer HPfB nucleosome positioned downstream. We identify direct physical interactions between HPfB and Rpo1N RNA polymerase subunit, establishing a defined polymerase-histone interface during transcription. Structural comparisons across defined elongation states demonstrate that RNA polymerase first translocates on DNA using the proximal histone dimer as an anchor, followed by destabilisation of the distal dimer. The DNA exiting the nucleosome is partially unwrapped and is redirected traversing the Rpo4/7 stalk. At extended transcript lengths nucleosome organisation is lost, indicating that transcription elongation ultimately disrupts histone-DNA interactions. This work provides the first structural insight into transcription through chromatin in archaea, revealing a mechanism in which the archaeal RNA polymerase actively remodels nucleosomes via DNA redirection and histone displacement, aided by direct polymerase-histone contacts.

molecular biology↗

bioRxiv2024

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.

biochemistry↗

bioRxiv2023

Uncovering the temporal dynamics and regulatory networks of thermal stress response in a hyperthermophile using transcriptomics and proteomics

Extremophiles, such as the hyperthermophilic archaeon Pyrococcus furiosus, thrive under extreme conditions and must rapidly adapt to changes in the physical parameters of their natural environment for short-term and long-term survival. When inhabiting hydrothermal vents, these organisms face substantial temperature gradients, necessitating the evolution of adaptive thermal stress mechanisms. However, the dynamics and coordination of cellular responses at the transcriptome and proteome levels remain underexplored. This study presents an integrated analysis of RNA-sequencing and mass spectrometry data to elucidate the transcriptomic and proteomic responses to heat and cold shock stress and recovery in P. furiosus. Our results reveal surprisingly rapid and dynamic changes in gene and protein expression patterns associated with these stress responses. Heat shock triggers extensive transcriptome reprogramming, orchestrated by the transcriptional regulator Phr, which targets a broader gene repertoire than previously demonstrated. For heat shock signature genes, RNA levels swiftly return to baseline upon recovery, while protein levels remain persistently upregulated, reflecting a rapid but more sustained response. Intriguingly, cold shock at 4{degrees}C elicits distinct short-term and long-term responses at both RNA and protein levels. By conducting a cluster analysis, we identified gene sets with either congruent or contrasting trends in RNA and protein changes. Notably, these clusters represent well-separated arCOG groups and appear to be tailored to their individual cellular responses. Our study provides a comprehensive overview of the cellular response to temperature stress, advancing our understanding of stress response mechanisms in hyperthermophilic archaea and provide valuable insights into the molecular adaptations that facilitate life in extreme environments.

microbiology↗

bioRxiv2020

CopR, a global regulator of transcription to maintain copper homeostasis in Pyrococcus furiosus

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analysed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterise the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 M copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the complex regulatory network that enables Pyrococcus to respond to increased copper concentrations.

microbiology↗