Expression, reconstitution, and structure of an archaeal RNA degrading exosome.
The exosome is a protein complex that participates in a wide variety of RNA processing, degradation, and quality-control pathways. The exosome is conserved in all eukaryotes studied to date and is also present in many archaeal organisms, albeit in a simpler form. To gain insights into the architecture of the exosome complex, we have chosen the hyperthermophilic archaeum Sulfolobus solfataricus as a model system. Here we describe the coexpression, purification, and crystal structure determination of archaeal exosome complexes. To understand how the archaeal exosome binds and degrades RNA, we designed RNA substrates for degradation experiments exploiting the knowledge of the geometric constraints of the exosome structure. Furthermore, we describe several crystal structures in which RNA substrates were diffused into crystals and how anomalous scattering from 5-iodo-uridine-modified RNA was used to locate low-occupancy RNA binding sites.
Lorentzen
Esben
E
Birkbeck College London, Institute of Structural Molecular Biology, London, United Kingdom.
Conti
Elena
E
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D055354:Exosomes / Q000235:genetics / Q000378:metabolism*; D019766:Genes, Archaeal; D011487:Protein Conformation; D019642:RNA, Archaeal / Q000378:metabolism*; D048229:Sulfolobus solfataricus / Q000235:genetics*
D019843:Archaeal Proteins; D019642:RNA, Archaeal