Tuck, L. R. | bioRxiv Science

bioRxiv2019

Start codon context controls translation initiation in the fungal kingdom

Eukaryotic protein synthesis initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but studies of S. cerevisiae suggest this context is of little importance in fungi. We tested this concept in two pathogenic Cryptococcus species by genome-wide mapping of translation and of mRNA 5 and 3 ends. We observed that upstream open reading frames (uORFs) are a major contributor to translation repression, that uORF use depends on the Kozak sequence context of its start codon, and that uORFs with strong contexts promote nonsense-mediated mRNA decay. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Further analysis shows that such dual-localization is also predicted to be common in Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that programs the expression and structures of proteins in fungi.

genomics↗

bioRxiv2018

Conservation of the structural and functional architecture of encapsulated ferritins in bacteria and archaea

Iron is an essential element for many biological processes; however, due to its high reactivity iron can also be very toxic, producing reactive oxygen species through Fenton chemistry. Ferritins protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing the resulting iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. We recently characterised the Rhodospirillum rubrum EncFtn, showing that although enzymatically active, due to its open structure it requires the association with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether the structure and catalytic activity is conserved across the family. Here we structurally characterise two EncFtn members from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the same distinct annular decamer topology observed in R. rubrum EncFtn, with the ferroxidase centre (FOC) formed between one of the dimer interfaces. Solution and native mass spectrometr analyses show that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The catalytic role of the EncFtn proteins was confirmed by biochemical assays, and we show that Zn(II) ions inhibit the ferroxidase activity of the EncFtn proteins to varying degrees. Our results represent a further step in the characterisation of the recently discovered EncFtn ferritin-like sub-family, indicating a common structural organisation and catalytic activity, despite diverse host environments.

biochemistry↗