Mammalian fatty acid synthase is a large multienzyme that catalyzes all steps of fatty acid synthesis. We have determined its crystal structure at 3.2 angstrom resolution covering five catalytic domains, whereas the flexibly tethered terminal acyl carrier protein and thioesterase domains remain unresolved. The structure reveals a complex architecture of alternating linkers and enzymatic domains. Substrate shuttling is facilitated by flexible tethering of the acyl carrier protein domain and by the limited contact between the condensing and modifying portions of the multienzyme, which are mainly connected by linkers rather than direct interaction. The structure identifies two additional nonenzymatic domains: (i) a pseudo-ketoreductase and (ii) a peripheral pseudo-methyltransferase that is probably a remnant of an ancestral methyltransferase domain maintained in some related polyketide synthases. The structural comparison of mammalian fatty acid synthase with modular polyketide synthases shows how their segmental construction allows the variation of domain composition to achieve diverse product synthesis.

SummaryProteins are synthesized by large molecular machines, the ribosomes. Ribosomes consist of two subunits of unequal size, the small subunit and the large subunit. The small subunit contains the decoding site, where the sequence information contained in the messenger RNA (mRNA) is translated into protein sequence, while the large subunit contains the peptidyl transferase center, which catalyzes the peptide bond formation between amino acids of the nascent protein. Translation initiation in eukaryotes requires twelve initiation factors in addition to the ribosome.The aim of this thesis was to determine the crystal structure of an initiation complex of the eukaryotic ribosome in order to make high-resolution structural information on eukaryotic ribosomes and their initiation complexes available. The complex of the Tetrahymena thermophila 40S ribosomal subunit with eukaryotic initiation factor 1 (eIF1) was crystallized in three space groups. Phasing of the dataset with tantalum bromide clusters and subsequent non-crystallographic symmetry multicrystal averaging resulted in electron density maps at 3.9Å that were of sufficient quality to build the entire structure. The resulting model comprises the entire 18S rRNA, 33 ribosomal proteins and initiation factor eIF1.The structure gives insights into the evolution of the eukaryotic ribosome, into signaling at the eukaryotic ribosome and into the function of eIF1 during initiation. The eukaryotic 40S ribosomal subunit contains more proteins that the bacterial 30S subunit, which are engaged in stronger protein-protein interactions and have replaced a bacterial rRNA feature at the beak. Expansion segments of the rRNA are clustered at the back of the 40S subunit. The signaling hub protein RACK1 is an integral part of the ribosome that contacts three ribosomal proteins (rpS3e, rpS16e and rpS17e). The phosphorylation site of ribosomal protein rpS6e, which is a downstream target of the mTOR pathway, is located at the end of a long C-terminal helix, which stretches from the bottom to the back of the 40S subunit. In Tetrahymena, rpS4e, which is directly adjacent to rpS6e, is phosphorylated instead.The study yielded insights into the structural basis of the ability of eIF1 to sense the start codon recognition during translation initiation. Initiation factor eIF1 was found bound to the 40S on top of helix 44, directly below the P site. In the structure eIF1 extends a basic loop into the mRNA channel and is therefore in principle able to sense the conformation of mRNA and tRNA by interaction with their phosphate backbone. The structure presented in this thesis is the first structure of an entire eukaryotic ribosomal subunit and will form the foundation for structure-guided studies of eukaryotic translation. 5 ZusammenfassungProteine werden von den Ribosomen synthetisiert. Ribosomen sind grosse molekulare

ARS-CoV-2 is the causing agent of the COVID-19 pandemic and belongs to the genus of beta-coronaviruses, with enveloped, positive sense and single-stranded genomic RNA 1. On entering host cells, the viral genomic RNA is translated by the cellular protein synthesis machinery to produce a set of non-structural proteins (NSPs) 2. NSPs render the cellular conditions favorable for viral infection and viral mRNA synthesis 3. Coronaviruses have evolved specialized mechanisms to hijack the host gene expression machinery and employ cellular resources to regulate viral protein production. Such mechanisms are common for many viruses and include inhibition of host protein synthesis and endonucleolytic cleavage of host messenger RNAs (mRNAs) 4,5. In cells infected with the closely related SARS-CoV, one of the most enigmatic viral proteins is the host shutoff factor, Nsp1. Nsp1 is encoded by the gene closest to the 5′ end of the viral genome and is among the first proteins to be expressed after cell entry and infection to repress multiple steps of host protein expression 6-9. Initial structural characterization of the isolated SARS-CoV Nsp1 protein revealed the structure of its N-terminal domain, whereas its C-terminal region was flexibly disordered 10. Furthermore, it was shown that SARS-CoV Nsp1 suppresses host innate immune functions, mainly by targeting type I interferon expression and antiviral signaling pathways 11. Taken together, Nsp1 serves as a potential virulence factor for coronaviruses and represents an attractive target for live attenuated vaccine development 12,13. To provide molecular insights into the mechanism of SARS-CoV-2 Nsp1-mediated translation inhibition, we solved the structures of ribosomal complexes isolated from HEK293 lysates supplemented with recombinant purified Nsp1 as well as of an in vitro reconstituted 40S-Nsp1 complex using cryo-EM. We complement our findings by reporting in vitro and in vivo translation inhibition in the presence of Nsp1 that is relieved after mutating key interacting residues. Furthermore, we show that the translation output of reporters containing full-length viral 5′ untranslated regions (UTRs) is significantly enhanced, which could explain how Nsp1 inhibits global translation while still translating sufficient amounts of viral mRNAs. Results C-terminal domain of SARS-CoV-2 Nsp1 binds to the mRNA entry channel. To elucidate the mechanism of how Nsp1 inhibits translation, we aimed to identify the structures of potential ribosomal complexes as binding targets (Fig. 1). Previously, it has been suggested that Nsp1 mainly targets the ribosome at the translation initiation step 9. We thus treated lysed HEK293E cells with bacterially expressed and purified Nsp1 and loaded the cleared lysate on a sucrose gradient. Fractions containing ribosomal particles were then analyzed for the presence of Nsp1. Interestingly, Nsp1 not only co-migrated with 40S particles, but also with 80S ribosomal complexes (Fig. 1c), suggesting that it interacts with a range of different ribosomal states. W...

Mammalian mitochondrial ribosomes (mitoribosomes) synthesize mitochondrially encoded membrane proteins that are critical for mitochondrial function. Here we present the complete atomic structure of the porcine 55S mitoribosome at 3.8 angstrom resolution by cryo-electron microscopy and chemical cross-linking/mass spectrometry. The structure of the 28S subunit in the complex was resolved at 3.6 angstrom resolution by focused alignment, which allowed building of a detailed atomic structure including all of its 15 mitoribosomal-specific proteins. The structure reveals the intersubunit contacts in the 55S mitoribosome, the molecular architecture of the mitoribosomal messenger RNA (mRNA) binding channel and its interaction with transfer RNAs, and provides insight into the highly specialized mechanism of mRNA recruitment to the 28S subunit. Furthermore, the structure contributes to a mechanistic understanding of aminoglycoside ototoxicity.

Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.