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Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins.sup.1,2. Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB1.sup.3. Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO1.sup.4. The final maturation events, including the activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model organism Saccharomyces cerevisiae. Studies of five cryo-electron microscopy structures reveal the composition and conformational progression in the final maturation events of human 40S ribosomal subunit assembly., Author(s): Michael Ameismeier [sup.1] , Ivo Zemp [sup.2] , Jasmin van den Heuvel [sup.2] [sup.3] , Matthias Thoms [sup.1] , Otto Berninghausen [sup.1] , Ulrike Kutay [sup.2] , Roland Beckmann [...]

Author(s): Paul Huter [1]; Claudia Mller [1]; Bertrand Beckert [1, 2]; Stefan Arenz [1]; Otto Berninghausen [1]; Roland Beckmann [1]; Daniel N. Wilson (corresponding author) [1, 2] In bacteria, ribosomes [...]

The biogenesis of secretory as well as transmembrane proteins requires the activity of the universally conserved protein-conducting channel (PCC), the Sec61 complex (SecY complex in bacteria). (1) In eukaryotic cells the PCC is located in the membrane of the endoplasmic reticulum where it can bind to translating ribosomes for cotranslational protein transport. The Sec complex consists of three subunits (Sec61α, β and γ) and provides an aqueous environment for the translocation of hydrophilic peptides as well as a lateral opening in the Sec61α subunit that has been proposed to act as a gate for the membrane partitioning of hydrophobic domains (2). A plug helix and a so-called pore ring are believed to seal the PCC against ion flow and are proposed to rearrange for accommodation of translocating peptides (2,3). Several crystal and cryo-electron microscopy structures revealed different conformations of closed and partially open Sec61 and SecY complexes (2,4-8). However, in none of these samples has the translocation state been unambiguously defined biochemically. Here we present cryo-electron microscopy structures of ribosome-bound Sec61 complexes engaged in translocation or membrane insertion of nascent peptides. Our data show that a hydrophilic peptide can translocate through the Sec complex with an essentially closed lateral gate and an only slightly rearranged central channel. Membrane insertion of a hydrophobic domain seems to occur with the Sec complex opening the proposed lateral gate while rearranging the plug to maintain an ion permeability barrier. Taken together, we provide a structural model for the basic activities of the Sec61 complex as a protein-conducting channel., Ribosome-protein-conducting channel (PCC) complexes were formed using a well-established in vitro protein translocation system: a wheat germ translation extract combined with canine pancreatic endoplasmic reticulum membranes (9). We chose nascent [...]

Protein synthesis in all cells is carried out by macromolecular machines called ribosomes. Although the structures of prokaryotic, yeast and protist ribosomes have been determined, the more complex molecular architecture of metazoan 80S ribosomes has so far remained elusive. Here we present structures of Drosophila melanogaster and Homo sapiens 80S ribosomes in complex with the translation factor eEF2, E-site transfer RNA and Stm1-like proteins, based on high-resolution cryo-electron-microscopy density maps. These structures not only illustrate the co-evolution of metazoan-specific ribosomal RNA with ribosomal proteins but also reveal the presence of two additional structural layers in metazoan ribosomes, a well-ordered inner layer covered by a flexible RNA outer layer. The human and Drosophila ribosome structures will provide the basis for more detailed structural, biochemical and genetic experiments., Crystal structures of prokaryotic ribosomal particles have provided insights into protein biosynthesis at both a structural and a functional level (1). In contrast to their bacterial counterparts, eukaryotic ribosomes are [...]

Ribosome-driven protein biosynthesis is comprised of four phases: initiation, elongation, termination and recycling. In bacteria, ribosome recycling requires ribosome recycling factor and elongation factor G, and several structures of bacterial recycling complexes have been determined. In the eukaryotic and archaeal kingdoms, however, recycling involves the ABC-type ATPase ABCE1 and little is known about its structural basis. Here we present cryoelectron microscopy reconstructions of eukaryotic and archaeal ribosome recycling complexes containing ABCE1 and the termination factor paralogue Pelota. These structures reveal the overall binding mode of ABCE1 to be similar to canonical translation factors. Moreover, the iron-sulphur cluster domain of ABCE1 interacts with and stabilizes Pelota in a conformation that reaches towards the peptidyl transferase centre, thus explaining how ABCE1 may stimulate peptide-release activity of canonical termination factors. Using the mechanochemical properties of ABCE1, a conserved mechanism in archaea and eukaryotes is suggested that couples translation termination to recycling, and eventually to re-initiation., Recycling of ribosomes for a new round of translation initiation is an essential part of protein synthesis. In archaea and eukaryotes recycling has been shown to require the highly conserved [...]

Swi2/Snf2-type ATPases regulate genome-associated processes such as transcription, replication and repair by catalysing the disruption, assembly or remodelling of nucleosomes or other protein-DNA complexes (1,2). It has been suggested that ATP-driven motor activity along DNA disrupts target protein-DNA interactions in the remodelling reaction (3-5). However, the complex and highly specific remodelling reactions are poorly understood, mostly because of a lack of high-resolution structural information about how remodellers bind to their substrate proteins. Mot1 (modifier of transcription 1 in Saccharomyces cerevisiae, denoted BTAF1 in humans) is a Swi2/Snf2 enzyme that specifically displaces the TATA box binding protein (TBP) from the promoter DNA and regulates transcription globally by generating a highly dynamic TBP pool in the cell (6,7). As a Swi2/Snf2 enzyme that functions as a single polypeptide and interacts with a relatively simple substrate, Mot1 offers an ideal system from which to gain a better understanding of this important enzyme family. To reveal how Mot1 specifically disrupts TBP-DNA complexes, we combined crystal and electron microscopy structures of Mot1-TBP from Encephalitozoon cuni-culi with biochemical studies. Here we show that Motl wraps around TBP and seems to act like a bottle opener: a spring-like array of 16 HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats grips the DNA-distal side of TBP via loop insertions, and the Swi2/Snf2 domain binds to upstream DNA, positioned to weaken the TBP-DNA interaction by DNA translocation. A 'latch' subsequently blocks the DNA-binding groove of TBP, acting as a chaperone to prevent DNA re-association and ensure efficient promoter clearance. This work shows how a remodelling enzyme can combine both motor and chaperone activities to achieve functional specificity using a conserved Swi2/Snf2 translocase., Motl is highly conserved among eukaryotes and consists of an amino-terminal TBP binding region of approximately 90-140 kDa with predicted HEAT repeats, followed by a carboxy-terminal Swi2/ Snf2-type ATPase domain [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.09.002 Byline: Alessandro Vannini (1), Rieke Ringel (1), Anselm G. Kusser (1), Otto Berninghausen (1), George A. Kassavetis (2), Patrick Cramer (1) Abstract: RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation. Author Affiliation: (1) Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universitat Munchen, Feodor-Lynen-Strasse 25, 81377 Munich, Germany (2) Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA Article History: Received 3 May 2010; Revised 6 July 2010; Accepted 11 August 2010 Article Note: (miscellaneous) Published: September 30, 2010

Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric C[O.sup.2] in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone [RbcX.sub.2]. RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by [RbcX.sub.2]. As revealed by the structure of a [RbcL.sub.8]-[([RbcX.sub.2]).sub.8] assembly intermediate, [RbcX.sub.2] acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates [RbcL.sub.8] core assembly. Finally, addition of RbcS results in [RbcX.sub.2] release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly., Rubisco is one of the most abundant enzymes in nature. It converts atmospheric carbon dioxide to organic compounds in photosynthesis (1), and thus has important implications in improving crop yield [...]