The Ski2-Ski3-Ski8 (SKI) complex is a conserved multi-protein assembly required for the cytoplasmic functions of the exosome, including messenger RNA (mRNA) turnover, surveillance and interference. The helicase Ski2, the tetratricopeptide repeat (TPR) protein Ski3 and the �-propeller Ski8 assemble in a heterotetramer with 1:1:2 stoichiometry. While the function of the Ski2-Ski3-Ski8 complex as a general cofactor of the cytoplasmic exosome has been well established, it remains largely unclear how it contributes to the regulation of the exosome. The PhD thesis at hand addresses this question by investigating the structural and biochemical properties of the Ski2-Ski3-Ski8 complex. Solving the crystal structure of the 113 kDa helicase region of S. cerevisiae Ski2 by experimental phasing revealed the presence of a canonical DExH core and an atypical accessory domain that is inserted in the helicase core. This insertion domain binds ribonucleic acid (RNA) unspeci�cally and is located at the RNA entry site of the helicase core. The overall architecture of Ski2 including the presence of an accessory domain is similar to the structure of the related helicase Mtr4, but the structural and biochemical properties of the accessory domains from both proteins are di�erent. The Ski2 insertion domain is not required for formation of the Ski2-Ski3-Ski8 complex. Its removal allowed to crystallize a Ski2�insert-Ski3-Ski8 complex from S. cerevisiae, and the crystal structure of this 370 kDa core complex was determined experimentally. It shows that Ski3 forms an array of 33 TPR motifs, creating a sca�old for the other subunits. Ski3 and the two Ski8 subunits bind the helicase core of Ski2 and position it centrally within the complex. This creates an extended internal RNA channel and modulates the enzymatic properties of the Ski2 helicase. Both Ski8 subunits are bound through a structurally conserved motif. A similar motif is present and functional in yeast Spo11, a protein that initiates deoxyribonucleic acid (DNA) double strand breaks during meiotic recombination. Association of Ski8 to either complex is mutually exclusive, rationalizing how Ski8 can perform its two distinct roles in mRNA metabolism and meiotic recombination. Biochemical studies suggest that the SKI complex can thread RNAs directly to the exosome, coupling the helicase and the exoribonuclease through a continuous channel of 43-44 nucleotides length. Finally, an internal regulatory mechanism in the Ski2-Ski3-Ski8 complex was identi�ed. Both the Ski2-insertion domain and the Ski3 N-terminus cooperate to inhibit ATPase and helicase activity of Ski2 when bound in the SKI complex. Thus, the SKI complex regulates exosome activity in two ways. First by a direct substrate channeling mechanism to the exosome that connects helicase and nuclease activities of both complexes which may activate the exosome towards certain substrates. Second, by an inhibitory mechanism that regulates substrate access to the helicase complex, which is a prerequisite for controlling the exosome's substrate speci�city. This doctoral thesis provides the �rst structural description of the entire yeast SKI complex and identi�es two mechanisms that may contribute to regulation of the activity of the cytoplasmic exosome.

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Apr 09, 2013

Carbon nitrides and carbon nitride derivatives are promising photocatalysts. The main focus of this thesis is the synthesis and characterization of various carbon nitrides (incompletely condensed melon, carbon nitride doped cesium titanate, ultra-long calcined melon, and OH-melem). Those carbon nitrides were then tested with regard to their photocatalytic properties. In the first part of chapter 3 of this thesis, we focus on a material called ‘‘melem oligomer’’. Two different synthesis routes were applied (open system and half open system) and the composition and structure of this material was studied. Melem with two different crystalline structures and some amorphous residues were found in the product. We also tested the photocatalytic activity of melem oligomer and confirmed hydrogen production from water with a relatively low rate of 2 μmol g-1 h-1. In the second part of chapter 3, we synthesized ultra-long calcined melamine which may have a morphology similar to the ‘‘g-C3N4 nanosheets’’. We analyzed both the composition and structure and investigated the efficiency of the presumed g-C3N4 nanosheets for hydrogen production from water. Ultra-long calcined melamine showed the best photoactivity which is twice that of melon at 490 °C. This is most likely due to the interesting morphology and high surface area. In chapter 4, melem oligomer was doped with cesium titanate in situ. Different calcination times were applied and various characterization techniques were used to investigate the composition, structure and morphology of the obtained materials. The efficiency of this hybrid photocatalyst for hydrogen production did not show higher photoactivity than the pure carbon nitrides except in the case of 16 h calcination which was the optimum calcination time overall. In chapter 5, OH-melem with a composition close to 2-oxo-6,10-diamino-s-heptazine, which could be a precursor of oxygen-doped g-C3N4, was synthesized and characterized by various techniques. Crystallinity is rather low in this oxygen containing species. NMR spectra differ from melem or cyameluric acid and XPS results confirm the presence of C=O groups. Overall, different carbon nitrides and carbon nitride derivatives were synthesized and chemically investigated to gain further knowledge on their synthesis, chemical properties and their resulting application as photocatalysts.

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Mar 25, 2013

The synthesis and properties of explosive urea and triazine derivatives is investigated on behalf of the explosive parameters and the full characterization of the molecules. (Chapter I-III) The class of oxadiazole derivatives is enhanced from the known explosive 1,2,5 oxadiazole (furazane) derivatives to the 1,2,4 oxadiazole derivatives. This molecule class is thoroughly investigated by all terms of chemical and explosive material matter and especially the 1,2,4-oxadiazol-5-one derivatives are compared to the corresponding tetrazole derivatives which were by far the most investigated molecule moiety of Prof. Dr. T.M. Klapoetke et al. for more than the last ten years. The 1,2,4 oxadiazol-5-one derivatives do only value as comparable model molecule to the tetrazole but were found to be good explosives themselves. So the triaminoguanidinium 1,2,4-oxadiazol-5-onate is suitable as low temperature propellant, the potassium and cesium 1,2,4-oxadiazol-5-onate are found to be good additions for NIR-flares and last but not least the best performing molecule was found to be the 3,5-diamino-1,2,4-oxadiazolium 5-aminotetrazolate, which combines the stability of the oxadiazole moiety with the very exothermic properties of a tetrazole in its best way. (Chapter IV-V) The 3-amino-1,2,4(4H)-oxadiazol-5-one is investigated thoroughly and detected to be a chemically and thermodynamically more stable system which can be functionalized according to methods known prior in the working group. The 3-dinitromethyl-1,2,4(4H)-oxadiazol-5-one is found a promising explosive class which can be combined as anion with a wide range of cations to tailor the stability and performance. The overall conclusion is that the 1,2,4-oxadiazole are chemical suitable as well as secondary explosives, propellants and pyrotechnics.

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Mar 21, 2013

DYNAMIC TRANSCRIPTOME ANALYSIS MEASURES RATES OF MRNA SYNTHESIS AND DECAY IN YEAST To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non-perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress-responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress-induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems.

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Feb 27, 2013