Structural and functional characterization of the yeast Ski2-Ski3-Ski8 complex

Halbach, Felix (2013): Structural and functional characterization of the yeast Ski2-Ski3-Ski8 complex. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

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.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: RNA degradation, helicase, x-ray structure, exosome
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 9. April 2013
• 1. Bericht­erstatter:in: Conti, Elena