Crystal structures of Rea1-MIDAS bound to its ribosome assembly factor ligands resembling integrin-ligand-type complexes

The Rea1 AAA(+)-ATPase dislodges assembly factors from pre-60S ribosomes upon ATP hydrolysis, thereby driving ribosome biogenesis. Here, we present crystal structures of Rea1-MIDAS, the conserved domain at the tip of the flexible Rea1 tail, alone and in complex with its substrate ligands, the UBL domains of Rsa4 or Ytm1. These complexes have structural similarity to integrin alpha-subunit domains when bound to extracellular matrix ligands, which for integrin biology is a key determinant for force-bearing cell-cell adhesion. However, the presence of additional motifs equips Rea1-MIDAS for its tasks in ribosome maturation. One loop insert cofunctions as an NLS and to activate the mechanochemical Rea1 cycle, whereas an additional beta-hairpin provides an anchor to hold the ligand UBL domains in place. Our data show the versatility of the MIDAS fold for mechanical force transmission in processes as varied as integrin-mediated cell adhesion and mechanochemical removal of assembly factors from pre-ribosomes

Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones

Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1’s WD-repeat β-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation

Structural studies on the Velvet-Complex from Aspergillus nidulans

Die Regulation des sekundären Metabolismus und der Entwicklung in Aspergillus nidulans sind zwei miteinander verbundene Prozesse. Ihre Regulation geschieht in Abhängigkeit von Licht. Die molekulare Grundlage dieser Verbindung ist ein Komplex bestehend aus den beiden Velvet-Proteinen VeA, VelB und der putativen Methyltransferase LaeA. Ein weiteres Protein der Velvet-Familie ist VosA, dass in Verbindung mit VelB eine essenzielle Rolle in der Sporogenese und Biosynthese von Trehalose spielt. Bisheriges Wissen über die generelle Funktion der einzelnen Proteine basiert überwiegend auf in vivo Analysen. Unbekannt hingegen ist die molekulare Funktionsweise sowie Struktur dieser Proteine. Zu diesem Zweck wurde in der vorliegenden Arbeit versucht dieses Defizit durch eine röntgenkristallographische Strukturanalyse der Proteine aufzuheben.The regulation of secondary metabolism and development in Aspergillus nidulans are two linked processes. Their regulation is dependent on light. The molecular basis for this connection is a complex composed of the two Velvet-proteins VeA, VelB and the putative methyltransferase LaeA (Velvet-complex). VelB together with VosA, which is another Velvet-protein, are essential for sporogenisis and biosynthesis of trehalose. Current knowledge about the general function of these proteins is based mostly on in vivo analysis. The molecular mode of function and structure of these proteins however is unknown. The current work aimed at resolving this by means of x-ray crystallographic structure analyis of these proteins

Correction: Mpp10 represents a platform for the interaction of multiple factors within the 90S pre-ribosome.

[This corrects the article DOI: 10.1371/journal.pone.0183272.]

Structural basis for 5'-ETS recognition by Utp4 at the early stages of ribosome biogenesis.

Eukaryotic ribosome biogenesis begins with the co-transcriptional assembly of the 90S pre-ribosome. The 'U three protein' (UTP) complexes and snoRNP particles arrange around the nascent pre-ribosomal RNA chaperoning its folding and further maturation. The earliest event in this hierarchical process is the binding of the UTP-A complex to the 5'-end of the pre-ribosomal RNA (5'-ETS). This oligomeric complex predominantly consists of β-propeller and α-solenoidal proteins. Here we present the structure of the Utp4 subunit from the thermophilic fungus Chaetomium thermophilum at 2.15 Å resolution and analyze its function by UV RNA-crosslinking (CRAC) and in context of a recent cryo-EM structure of the 90S pre-ribosome. Utp4 consists of two orthogonal and highly basic β-propellers that perfectly fit the EM-data. The Utp4 structure highlights an unusual Velcro-closure of its C-terminal β-propeller as relevant for protein integrity and potentially Utp8 recognition in the context of the pre-ribosome. We provide a first model of the 5'-ETS RNA from the internally hidden 5'-end up to the region that hybridizes to the 3'-hinge sequence of U3 snoRNA and validate a specific Utp4/5'-ETS interaction by CRAC analysis

DENR–MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation

<div><p>The succession of molecular events leading to eukaryotic translation reinitiation—whereby ribosomes terminate translation of a short open reading frame (ORF), resume scanning, and then translate a second ORF on the same mRNA—is not well understood. Density-regulated reinitiation and release factor (DENR) and multiple copies in T-cell lymphoma-1 (MCTS1) are implicated in promoting translation reinitiation both in vitro in translation extracts and in vivo. We present here the crystal structure of MCTS1 bound to a fragment of DENR. Based on this structure, we identify and experimentally validate that DENR residues Glu42, Tyr43, and Tyr46 are important for MCTS1 binding and that MCTS1 residue Phe104 is important for tRNA binding. Mutation of these residues reveals that DENR-MCTS1 dimerization and tRNA binding are both necessary for DENR and MCTS1 to promote translation reinitiation in human cells. These findings thereby link individual residues of DENR and MCTS1 to specific molecular functions of the complex. Since DENR–MCTS1 can bind tRNA in the absence of the ribosome, this suggests the DENR–MCTS1 complex could recruit tRNA to the ribosome during reinitiation analogously to the eukaryotic initiation factor 2 (eIF2) complex in cap-dependent translation.</p></div

High-resolution structure of MCTS1 binding an N-terminal fragment of DENR.

<p>(A) Domain architecture of DENR, MCTS1, and the related eIF2D (ligatin) protein. (B) A DENR truncation series identifies aas 24–51 of DENR as the minimum peptide capable of binding MCTS1 in <i>Escherichia coli</i>. Summary of data presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005160#pbio.2005160.s001" target="_blank">S1 Fig</a>. (C) Crystal structure of the minimal DENR–MCTS1 complex. MCTS1 contains an N-terminal DUF1947 (light green) and a C-terminal PUA domain (green). The DENR peptide (light orange) binds along the interface between the N- and C-terminal MCTS1 domains. Anomalous density (14σ), calculated with ANODE, is shown as a mesh. (D) DENR contains a zinc finger at the N-terminus, comprised of Cys34, 37, and 44. Although our DENR construct lacks a fourth residue (Cys53) to complete the Zn²⁺ coordination, the zinc finger is properly folded, as it is completed by His58 of a crystallographically related MCTS1 molecule. aa, amino acid; DENR, density-regulated reinitiation and release factor; DUF1947, domain of unknown function 1947; eIF1, eukaryotic initiation factor 1; eIF2D, eukaryotic initiation factor 2D; MCTS1, multiple copies in T-cell lymphoma-1; MDM2, mouse double minute 2; PUA, pseudouridine synthase and archaeosine transglycosylase; SUI1, suppressors of initiation codon mutations 1; SWIB <i>SWItch</i>/Sucrose Nonfermentable complex B; WH, winged helix</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p