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Büttner, Katharina (2007): Part I: Structural framework for the mechanism of archaeal exosomes in RNA processing; Part II: Structural insights into DNA duplex separation by the archaeal superfamily 2 helicase Hel308. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Part I The exosome is a conserved 3´- 5´ exoribonuclease complex involved in cellular RNA metabolic processes in eukaryotes and archaea. Its involvement in the accurate processing of nuclear RNA precursors and in the degradation of RNA in both nucleus and cytoplasm implies a central function in the eukaryotic RNA surveillance machinery. This widespread function implies the ability of the exosome to distinguish between RNA substrates that should be matured by the removal of nucleotides to a precisely defined end point, and defective RNAs that undergo rapid and complete degradation. However, the structural and molecular mechanisms of processive 3´- 5´ RNA degradation and substrate specificity remain unclear. To obtain insights into the structural and functional organization of the exosome, I determined crystal structures of two 230 kDa nine subunit exosome isoforms from Archaeoglobus fulgidus. Both exosome isoforms contain a hexameric ring of RNase PH-like domain subunits Rrp41 and Rrp42 with a central chamber. A trimer of Rrp4 or Csl4 subunits is situated on one side of the RNase PH domain ring and forms a multidomain macromolecular interaction surface with central S1 domains and peripheral KH and zinc-ribbon domains. Tungstate soaks identified three phosphorolytic active sites inside the central processing chamber. Additional structural and functional results suggest that the S1 domains of Rrp4 or Csl4 subunits and a subsequent neck in the RNase PH domain ring form an RNA entry pore that only allows access of unstructured RNA to the active sites. The structural results presented here can not only mechanistically unify observed features of exosomes, including processive 3´ RNA degradation of unstructured RNA, the requirement for regulatory factors and coactivators to degrade structured RNA, and the precision in processing RNA species to a defined length. But the high conservation of the archaeal exosome to the eukaryotic exosome and its additional high structural similarity to bacterial mRNA-degrading PNPase suggest a common basis for 3´ RNA-degradation in all kingdoms of life. Furthermore, the structure of the archaeal exosome reveals remarkable architectural and functional similarities to the protein degrading proteasome. Part II Adenosine triphosphate (ATP) dependent nucleic acid unwinding by superfamily 2 (SF2) helicases is required for numerous biological processes, including DNA recombination, RNA decay and viral replication. The structural and molecular mechanism for processive duplex unwinding of SF2 helicases is still unclear, in part due to a lack of structural insights into the actual strand separation reaction. Archaeal SF2 helicase Hel308 preferentially unwinds lagging strands at replication forks and is closely sequence related to human PolΘ and Hel308 as well as Drosophila Mus308. Furthermore, the RecA ATPase-core of archaeal Hel308 shares high sequence conservation to the SF2 RNA decay factors Ski2p and Mtr4p. Thus, archaeal Hel308 appears as representative model to understand processive 3´- 5´ DNA unwinding by SF2 helicases. During this PhD thesis crystal structures of Archaeogloubs fulgidus Hel308 (afHel308) in the absence and presence of a 15mer duplex DNA containing a 10mer 3´-overhang were determinded using X-ray crystallography. afHel308 exhibits two typical SF2 RecA-like domains at the N-terminus. The C-terminus comprises a winged-helix (WH) domain, followed by a unique seven-helix-bundle domain and a helix-loop-helix (HLH) domain. The DNA bound structure captures the initial duplex separation and argues that initial strand separation does not require ATP binding. Comparison with ATP bound SF2 enzymes suggests that ATP binding and hydrolysis promotes processive unwinding of one base pair by a ratchet like transport of the 3’ product strand. In addition, the structure suggests that unwinding is promoted by a prominent β-hairpin loop. The identification of similar β-hairpin loops in Hepatitis C virus (HCV) NS3 helicase and RNA decay factors Ski2p and Mtr4p, and consistency of the results with biochemical data on HCV NS3 helicase argue that the observed duplex unwinding mechanism is applicable to a broader subset of processive SF2 helicases. Furthermore, the interaction between afHel308 and its DNA substrate also may explain how afHel308 is targeted to branched nucleic acid substrates.The presented results provide a first structural framework for duplex unwinding by processive SF2 helicases and reveal important mechanistic differences to SF1 helicases and the SF2 helicase RecG.