A Snu114-GTP-Prp8 module forms a relay station for efficient splicing in yeast

The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114-Prp8 complex co-purified with endogenous GTP. Snu114 exhibited weak, intrinsic GTPase activity that was abolished by the Prp8 Snu114-binding region. Exchange of GTP-contacting residues in Snu114, or of Prp8 residues lining the Snu114 GTP-binding pocket, led to temperature-sensitive yeast growth and affected the same set of splicing events in vivo. Consistent with dynamic Snu114-mediated protein interactions during splicing, our results suggest that the Snu114-GTP-Prp8 module serves as a relay station during spliceosome activation and disassembly, but that GTPase activity may be dispensable for splicing

Crystal Structure Analysis Reveals Functional Flexibility in the Selenocysteine-Specific tRNA from Mouse

Selenocysteine tRNAs (tRNA(Sec)) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec)-interacting factors are incompletely understood.We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec). tRNA(Sec) lacking the single-stranded 3'-acceptor end ((ΔGCCA)RNA(Sec)) yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCA)RNA(Sec) resembles the structure of human tRNA(Sec) determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec) used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Sec)in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+)-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec).We provide the most highly resolved structure of a tRNA(Sec) molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec) support its interaction with proteins

Kristallstrukturanalyse von Selenocystein-Biosynthese-Komponenten

Einige Organismen aus allen drei Domänen des Lebens Eukaryoten, Bakterien und Archaea - bauen Selenocystein (Sec) in Teile ihrer Proteine ein. Die Sec-Synthese findet an ihrer verwandten tRNASec statt, die zuerst mit Serin von der seryl-rRNAse-Synthase (SerRS) aminoacetyliert wird. Für die Umwandlung von Ser-tRNASec zu Sec-tRNASec in Bakterien ist die Selenocystein Synthase (SelA) verantwortlich, in Eukaryoten und Archaea sind es O-phospho-L-seryl-tRNASec kinase (PSTK) und Selenocystein Synthase (SecS). In Eukaryoten und Archaea ist SecS das letzte Enzym in der Sec Biosynthese. Es konvertiert O-phospho-L-seryl-tRNASec in Selenocysteyl-tRNASec unter Verwendung von Selenphosphat als Selen Donor. Die Kristallstruktur eines Fragmentes von 450 Resten des SecS aus Maus, das immer noch volle enzymatische Aktivität zeigten, wurde bei 1.65Å Auflösung gelöst. Das Protein ist aus der Familie der Faltungstyp I Familie der Pyridoxal-Phoshat (PLP)-Abhängigen Enzymen. Es besteht aus 3 Domänen die eine Nummer an Eigenschaften aufweisen, die bei keinem Enzym der Familie bisher gefunden wurde. Zwei SecS-Monomere interagieren eng miteinander und bilden zwei identische aktive Zentrem um einen PLP Cofaktor. Die Protomere tauschen gegenseitig ein langes SecS-spezifisches Insert der ersten Domäne, was das aktive Zentrum, verglichen mit anderen Faltungstyp I Enzymen, ändert. Zwei SecS Dimere bilden zusammen ein Homotetramer über die N-terminale Region, die einzigartig ist unter den SecS Orthologen. Ein detailierter Rekationsmechanismus für das Enzym basierend auf Aktivitätsprofielen von Inhibitoren wurde formuliert. tRNASec ist ein Schlüsselmolekül, das an allen Schritten des Biosyntheseweges beteiligt ist, in der Sec-Biosynthese und im Sec-Einbau. Um gut streuende Kristalle züchten zu können wurden rationelle Mutationen in murine tRNASec eingefügt. Kristallstrukturen von tRNASec ohne die Nukleotide 72-76 der Haarnadelstruktur des Akzeptors wurden bis zu einer Auflösung von 2.0 Å gelöst. Die Strukturen sind grob betrachtet identisch mit der humanen tRNASec die schon früher publiziert wurde. Unsere Struktur hingegen zeigt Details, die in der humanen Struktur bei 3.1Å verborgen blieben. Strukturelle Vergleiche der tRNASec Moleküle zeigte, dass der variable Arm und der Teil der Anticodon Haarnadelstruktur flexible Regionen sind, während hingegen der innere Teil des Moleküls steif ist. Möglicherweise spielen die flexiblen Teile der tRNASec eine Rolle bei der Kontaktherstellung mit interagierenden Proteinen. Die Anticodon-Schleifen von zwei tRNASec in der assymmetrischen Einheit nahmen eine 5- und 3- Nukleotid-Konformation ein. Wir kommen, basierend auf der Erstellung eines Modell von 2 -O-hydroxymethyierter Ribose an U34, zu dem Schluss, dass gänzlich modifizierte tRNASec die 5-Nukleotid Antikodon-Schleifen-Konformation favorisiert. Wassermoleküle 195 wurden lokalisiert, die sowohl die funktionalen Gruppen der Basen, als auch das Rückgrat kontaktieren. Interessanterweise sind Guanosine öfter hydriert als andere tRNASec Basen, was eventuell eine einzig dekorative chemische Funktionalität reflektiert. Soaking Experimente mit den Kristallen der tRNASec mit Mn2+ zeigen fünf putative Mg2+ bindestellen im Molekül. Alle gefundenen Metalle waren in Kontakt mit N7 Atomen von Guanosinen. Dennoch haben die Metallionen keine spezielle die 3D Struktur der tRNASec stabilisierende Funktion

Structure and Catalytic Mechanism of Eukaryotic Selenocysteine Synthase

In eukaryotes and Archaea, selenocysteine synthase (SecS) converts O-phospho-L-seryl-tRNA[Ser]Sec into selenocysteyltRNA[Ser]Sec using selenophosphate as the selenium donor compound. The molecular mechanisms underlying SecS activity are presently unknown. We have delineated a 450-residue core of mouse SecS, which retained full selenocysteyl-tRNA[Ser]Sec synthesis activity, and determined its crystal structure at 1.65Å resolution. SecS exhibits three domains that place it in the fold type I family of pyridoxal phosphate (PLP)-dependent enzymes. Two SecS monomers interact intimately and together build up two identical active sites around PLP in a Schiff-base linkage with lysine 284. Two SecS dimers further associate to form a homotetramer. The N terminus, which mediates tetramer formation, and a large insertion that remodels the active site set SecS aside from other members of the family. The active site insertion contributes to PLP binding and positions a glutamate next to the PLP, where it could repel substrates with a free α-carboxyl group, suggesting why SecS does not act on free O-phospho- L-serine. Upon soaking crystals in phosphate buffer, a previously disordered loop within the active site insertion contracted to form a phosphate binding site. Residues that are strictly conserved in SecS orthologs but variant in related enzymes coordinate the phosphate and upon mutation corrupt SecS activity. Modeling suggested that the phosphate loop accommodates the γ-phosphate moiety of OL-seryltRNA[Ser]Sec and, after phosphate elimination, binds selenophosphate to initiate attack on the proposed aminoacrylyl-tRNA[Ser]Sec intermediate. Based on these results and on the activity profiles of mechanism-based inhibitors, we offer a detailed reaction mechanism for the enzyme

Anticodon loop conformations in mouse tRNA^Sec.

<p>Stereo stick model of the closed anticodon loop conformation of molecule A (<b>A</b>) and of the open anticodon loop conformation of molecule B (<b>B</b>). The 2′-oxygen of the U34 ribose in molecule A that is methylated in a subset of cellular tRNA^Sec is shown as a thicker stick. Water molecules – cyan spheres. Hydrogen bonds are indicated by dashed lines.</p

Non-denaturing RNA purification.

<p>(<b>A</b>) Elution profile of <i>in vitro</i> transcribed mouse tRNA^Sec from a MonoQ column. Peak 1 – unincorporated rNTPs, T7 RNA polymerase and other proteins; Peak 2 – abortive synthesis transcripts; Peak 3 – desired RNA sample; Peak 4 – aggregates or higher molecular weight nucleic acids. The gradient (buffer B from 30 to 100%) is shown as a dashed line. (<b>B</b>) Denaturing SDS PAGE analysis of peak fractions from Peaks 1–3. T7 RNA polymerase and molecular weight markers (M) were loaded as references. Protein bands were stained with Coomassie. (<b>C</b>) Denaturing urea PAGE analysis of peak fractions eluted from the MonoQ column. S – crude transcription extract. RNA bands were stained with methylene blue. (<b>D</b>) Elution profile of mouse tRNA^Sec from a Superdex 75 10/300 GL column.</p

tRNA^Sec constructs for crystallization screening.

<p>RNA 1 represents full-length tRNA^Sec. Canonical tRNA numbering was used throughout. Additional nucleotides (labeled with lower case Latin characters) and gaps (missing numbers) compared to the canonical tRNA numbering are indicated only in the scheme of RNA 1. RNAs 2 and 3 were created by site-directed mutagenesis and contain a UUCG (red) or a kissing loop (green) in place of the wt variable loop, respectively. Using the initial, mutated constructs, further DNA templates were generated for <i>in vitro</i> transcription, which allowed synthesis of tRNA^Sec species with deletion of the 3′-GCCA end (RNAs 4, 5 and 6) or with substitution of the 3′-GCCA with a self-complementary 3′-GCGC overhang (RNAs 7, 8 and 9).</p

Hydration and metal ion binding of mouse tRNA^Sec.

<p>(<b>A</b>) Hydration of the G27•U43 wobble bps in molecules A (carbon – gold) and B (carbon – silver) of mouse tRNA^Sec, showing a full complement of first-shell water molecules (cyan spheres). Hydrogen bonds are indicated by dashed lines. (<b>B</b>) Anomalous difference Fourier map contoured at the 5 σ level (green mesh) calculated with anomalous differences recorded from a Mn²⁺-soaked crystal and phases obtained from molecular replacement with the native structure as a search model. Molecule A – gold; molecule B – silver. There are three common Mn²⁺ sites (1–3) in the two tRNA^Sec molecules. Sites 4 and 5 were found only in chain B. The boxed region is shown in a close-up view in the following panel. (<b>C</b>) Close-up of the boxed region of panel (B). Mn²⁺ ion (site 4; purple sphere) apparently reinforcing the interaction of U9 (AD-linker) with A48•C45 (first bp of the variable arm).</p

Analysis of purified RNA.

<p>(<b>A</b>) Native and (<b>B</b>) denaturing PAGE analysis of fractions collected from Superdex 75 10/300 GL gel filtration. S – concentrated RNA sample after anion exchange chromatography. (<b>C</b>) Analysis of mouse tRNA^Sec by multi-angle static light scattering. The optical density at 260 nm (OD₂₆₀; magenta), Rayleigh ratio (Rq; blue) and differential refractive index (RI; yellow) were monitored during analytical gel filtration on a Superdex 200 10/300 GL column. The measurement was done by Wyatt Technology Europe GmbH.</p

Tertiary interactions of the AD-linker and variable arm.

<p>(<b>A</b>) Water-mediated interaction of the AD-linker with the first bp of the variable arm in mouse tRNA^Sec (molecule A – gold; molecule B – silver). Water molecules – cyan spheres. Hydrogen bonds are shown as dashed lines. (<b>B</b>) Close-up of the variable arm (molecule A). Nucleotides of the variable loop are labeled. (<b>C</b>) Superposition of the mouse tRNA^Sec variable loop (carbon – gold) with a stable GAGA tetraloop from the 23S rRNA sarcin/ricin domain (carbon – cyan; PDB ID 483D).</p