The RNA chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal architecture of the Hfq hexamer presents three well-characterized surfaces that allow it to bind sRNAs to stabilize them and engage target transcripts. Hfq-interacting sRNAs are categorized into two classes based on the surfaces they use to bind Hfq. By characterizing a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen (N) starvation, we corroborated the important role of the three RNA-binding surfaces for Hfq function. We uncovered two, previously uncharacterized, conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfq's RNA-binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilization of both sRNA classes, to the same extent as seen in bacteria devoid of Hfq. However, the alanine substitutions at these residues resulted in only modest alteration in stability and structure of Hfq. We propose that V22 and G34 have impact on Hfq function, especially critical under cellular conditions when there is an increased demand for Hfq, such as N starvation.
- MeSH
- bakteriální RNA * metabolismus genetika chemie MeSH
- dusík metabolismus MeSH
- Escherichia coli * genetika metabolismus MeSH
- hydrofobní a hydrofilní interakce * MeSH
- konzervovaná sekvence MeSH
- malá nekódující RNA * metabolismus genetika chemie MeSH
- mutace MeSH
- protein hostitelského faktoru 1 * metabolismus genetika chemie MeSH
- proteiny z Escherichia coli * metabolismus genetika chemie MeSH
- regulace genové exprese u bakterií MeSH
- stabilita RNA * genetika MeSH
- stanovení celkové genové exprese MeSH
- transkriptom genetika MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/.
Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double-strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C-terminal domain, known as the SAP domain. Using single-particle cryo-electron microscopy, mass spectrometric analysis of intermolecular cross-linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA-bound state. The second position, which was observed in both apo and DNA-bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA. DATABASES: EM maps have been deposited in EMDB (EMD-11933). Coordinates have been deposited in Protein Data Bank (PDB 7AXZ). Other data are available from corresponding authors upon a request.
- MeSH
- antigen Ku chemie MeSH
- DNA chemie MeSH
- dvouřetězcové zlomy DNA * MeSH
- konformace proteinů MeSH
- lidé MeSH
- oprava DNA spojením konců * MeSH
- proteinové domény MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although multiprotein membrane complexes play crucial roles in bacterial physiology and virulence, the mechanisms governing their quality control remain incompletely understood. In particular, it is not known how unincorporated, orphan components of protein complexes are recognised and eliminated from membranes. Rhomboids, the most widespread and largest superfamily of intramembrane proteases, are known to play key roles in eukaryotes. In contrast, the function of prokaryotic rhomboids has remained enigmatic. Here, we show that the Shigella sonnei rhomboid proteases GlpG and the newly identified Rhom7 are involved in membrane protein quality control by specifically targeting components of respiratory complexes, with the metastable transmembrane domains (TMDs) of rhomboid substrates protected when they are incorporated into a functional complex. Initial cleavage by GlpG or Rhom7 allows subsequent degradation of the orphan substrate. Given the occurrence of this strategy in an evolutionary ancient organism and the presence of rhomboids in all domains of life, it is likely that this form of quality control also mediates critical events in eukaryotes and protects cells from the damaging effects of orphan proteins.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- endopeptidasy chemie metabolismus MeSH
- membránové proteiny metabolismus MeSH
- proteinové domény MeSH
- proteolýza MeSH
- Shigella sonnei enzymologie metabolismus MeSH
- substrátová specifita MeSH
- transport elektronů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Susceptibility to prion diseases is largely determined by the sequence of the prion protein gene (PRNP), which encodes the prion protein (PrP). The recent emergence of chronic wasting disease (CWD) in Europe has highlighted the need to investigate PRNP gene diversity in European deer species, to better predict their susceptibility to CWD. Here we report a large genetic survey of six British deer species, including red (Cervus elaphus), sika (Cervus nippon), roe (Capreolus capreolus), fallow (Dama dama), muntjac (Muntiacus reevesii), and Chinese water deer (Hydropotes inermis), which establishes PRNP haplotype and genotype frequencies. Two smaller data sets from red deer in Norway and the Czech Republic are also included for comparison. Overall red deer show the most PRNP variation, with non-synonymous/coding polymorphisms at codons 98, 168, 226 and 247, which vary markedly in frequency between different regions. Polymorphisms P168S and I247L were only found in Scottish and Czech populations, respectively. T98A was found in all populations except Norway and the south of England. Significant regional differences in genotype frequencies were observed within both British and European red deer populations. Other deer species showed less variation, particularly roe and fallow deer, in which identical PRNP gene sequences were found in all individuals analysed. Based on comparison with PRNP sequences of North American cervids affected by CWD and limited experimental challenge data, these results suggest that a high proportion of wild deer in Great Britain may be susceptible to CWD.
- MeSH
- genetická predispozice k nemoci MeSH
- náchylnost k nemoci veterinární MeSH
- polymorfismus genetický * MeSH
- prionová bílkovina genetika MeSH
- sekvenční analýza DNA veterinární MeSH
- syndrom chronického chřadnutí genetika MeSH
- vysoká zvěř * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Norsko MeSH
- Spojené království MeSH
Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new-flagellum and the old-flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new-flagellum daughter in particular re-modelling rapidly and extensively in early G1. This re-modelling process involves an increase in cell body, flagellum and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old-flagellum daughter undergoes a different G1 re-modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that the two daughters of a proliferative division of T. brucei are non-equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non-equivalence.
- MeSH
- buněčné dělení MeSH
- cytokineze MeSH
- flagella genetika metabolismus MeSH
- proliferace buněk MeSH
- protozoální proteiny genetika metabolismus MeSH
- stadia vývoje MeSH
- Trypanosoma brucei brucei cytologie genetika růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
