Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits1. A multitude of transient assembly factors regulate and chaperone the systematic folding of preribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 Å. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix-domain proteins and their binding partners form a ring-like structure at ribosomal RNA (rRNA) domain boundaries to support the architecture of the maturing particle. The existence of mutually exclusive conformations of these pre-60S particles suggests that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly. These structures rationalize previous genetic and biochemical data and highlight the mechanisms that drive eukaryotic ribosome assembly in a unidirectional manner.

During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the yeast co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality-control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel and, together with ribosomal proteins, cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus.

sulfur-containing metabolites directly from the diet, plants, fungi, and bacteria are able to 43 assimilate and utilize sulfur from organic and inorganic sources (Barton, 2005). Sulfate (SO 4 2-) is 44 the most abundant source of sulfur in the environment and its utilization is contingent upon its 45 . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/131318 doi: bioRxiv preprint first posted online Apr. 26, 2017; 3 entry into the cell (Kertesz, 2000). In certain fungi, and prokaryotes, once internalized, SO 4 2-is 46 first reduced to sulfite (SO 3 2-), and then further to sulfide (S 2-), a form that can be used by the 47 cell (Kredich, Hulanicka, & Hallquist, 1979) (Fig. 1-S1). In Escherichia coli and other gram-48 negative bacteria, the culmination of the aforementioned sulfate assimilatory (also known as 49 reductive) pathway is the formation of cysteine by the addition of S 2-to O-acetylserine by 50 cysteine synthase, followed by the synthesis of methionine from homocysteine (Kredich, 1971) 51 ( Fig. 1-S1). Huang, 2014). The cysZ gene owes its name to its presence in the cysteine biosynthesis regulon. 64In two reports from thirty years ago, an E. coli K12 strain with a cysZ deletion showed a severe 65 impairment in its ability to accumulate SO 4 2-and was not viable in sulfate-free media without an 66 alternate sulfur source such as thiosulfate (S 2 O 3 2-) Parra, Britton, Castle, 67 Jones- . Recently, a third report studying the functional properties 68 . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/131318 doi: bioRxiv preprint first posted online Apr. 26, 2017; 4 of CysZ, concluded that the protein from E. coli functions as a high affinity, highly specific pH-69 dependent SO 4 2-transporter, directly regulated by the toxic, assimilatory pathway intermediate, 70 SO 3 2- (Zhang et al., 2014 RESULTS 93 Structure determination of CysZ 94Following a structural genomics approach aimed at crystallization for structural analysis, we 95 cloned and screened a total of 63 different bacterial homologs of CysZ for high-level expression 96 and stability in detergents (Love et al., 2010;Mancia & Love, 2011 The hexameric structure of PdCysZ 115The refined structure of PdCysZ comprises an entire hexamer of near-perfect D3 symmetry. 116Antiparallel pairs of protomers arrange together as a trimer of dimers (Fig. 1a), with the three- The CysZ protomer is an alpha-helical integral membrane protein with two long transmembrane 132 (TM) helices (H2b and H3a) and two pairs of shorter helices (H4b-H5a and H7-H8) that insert 133 only partially into the membrane (hemi-penetrating), forming a funnel or tripod-like shape within 134 the membrane (Fig. 2a, b). The protein has an extra-membranous hydrophilic 'head', comprising 135 an iris-like a...

Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.

During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel, and together with ribosomal proteins cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus.