Biology subjects

Koubek, J.

Publications and source records attributed to Koubek, J..

4 recordsLinked to original sources

bioRxiv2025

Structural basis for co-translational assembly of homo-oligomeric proteins

The efficient formation of protein complexes is essential for cellular function. Across all kingdoms of life, protein complexes frequently assemble co-translationally, either when one subunit is fully synthesized before interaction with nascent partner subunits (co-post) or when multiple nascent subunits interact co-translationally (co-co), with homomeric proteins particularly enriched among co-co assembling complexes. However, it is unknown whether co-co assembly of homomers occurs on the same mRNA in cis or across neighbouring mRNAs in trans, and how ribosomes spatially organize to enable co-co assembly. Using E. coli homodimeric protein PheA as a proof-of-concept model, we show by ribosome profiling and cryo-EM structural analysis that it employs the co-co assembly route. Co-co assembly of PheA is facilitated by the proximity of polypeptide exit tunnels, but does not rely on fixed ribosomal orientations. Surprisingly, PheA was identified as an exceptional candidate where trans-assembly is potentially the primary mode of PheA co-co assembly, generating large polysomal networks for PheA synthesis. Cis-assembly of PheA was less frequent and occurred mainly between non-adjacent ribosomes on the mRNA due to spatial restraints imposed by the arrangement of directly neighbouring ribosomes in polysomes. These findings reveal fundamental principles of how cells structurally organise fast and efficient co-translational protein complex assembly.

molecular biology↗

bioRxiv2025

Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response

The heat shock response (HSR) is the major defense mechanism against proteotoxic stress in the cytosol and nucleus of eukaryotic cells. Initiation and attenuation of the response are mediated by stress-dependent regulation of heat shock transcription factors (HSFs). Saccharomyces cerevisiae encodes a single HSF (Hsf1), facilitating the analysis of HSR regulation. Hsf1 is repressed by Hsp70 chaperones under non-stress conditions, and becomes activated under proteotoxic stress, directly linking protein damage and its repair to the HSR. J-domain proteins (JDPs) are essential for targeting of Hsp70s to their substrates, yet the specific JDP(s) regulating Hsf1 and connecting protein damage to HSR activation remain unclear. Here we show that the yeast nuclear JDP Apj1 primarily controls the attenuation phase of the HSR by promoting Hsf1s displacement from heat shock elements in target DNA. In apj1{Delta} cells, HSR attenuation is significantly impaired. Additionally, yeast cells lacking both Apj1 and the major JDP Ydj1 exhibit increased HSR activation even in non-stress conditions, indicating their distinct regulatory roles. Apj1s role in both nuclear protein quality control and Hsf1 regulation underscores its role in directly linking nuclear proteostasis to HSR regulation. Together these findings establish the nucleus as key stress-sensing signaling hub.

molecular biology↗

bioRxiv2025

A simple, fast and cost-efficient protocol for ultra-sensitive ribosome profiling

Ribosome profiling has become an essential tool for studying mRNA translation in cells with codon-level resolution. However, its widespread application remains hindered by the labour-intensive workflow, low efficiency and high costs associated with sequencing sample preparation. Here, we present a new cost-effective and ultra-sensitive library preparation method that significantly advances the applicability of ribosome profiling. By implementing bead-coupled enzymatic reactions and product purifications, our approach increases both yield and throughput while maintaining high reproducibility. Demonstrating the sensitivity of the protocol we prepared libraries from as little as 12 fmol of RNA, which expands the feasibility of ribosome profiling from minimal input samples, such as derived from small populations, stressed cells, or patient-derived specimens. Additionally, we validate the versatility of the protocol across multiple species and demonstrate its applicability for RNA-seq library preparation. Altogether, this protocol provides a highly accessible and efficient alternative to existing ribosome profiling workflows, facilitating research in previously challenging experimental contexts.

systems biology↗

bioRxiv2021

Cellular translational enhancer elements that recruit eukaryotic initiation factor 3

Translation initiation is a highly regulated process which broadly affects eukaryotic gene expression. Eukaryotic initiation factor 3 (eIF3) is a central player in canonical and alternative pathways for ribosome recruitment. Here we have investigated how direct binding of eIF3 contributes to the large and regulated differences in protein output conferred by different 5'-untranslated regions (5'-UTRs) of cellular mRNAs. Using an unbiased high-throughput approach to determine the affinity of budding yeast eIF3 for native 5'-UTRs from 4,252 genes, we demonstrate that eIF3 binds specifically to a subset of 5'-UTRs that contain a short unstructured binding motif, AMAYAA. eIF3 binding mRNAs have higher ribosome density in growing cells and are preferentially translated under certain stress conditions, supporting the functional relevance of this interaction. Our results reveal a new class of translational enhancer and suggest a mechanism by which changes in core initiation factor activity enact mRNA-specific translation programs.

systems biology↗