Abstract
The exosome is a conserved eukaryotic enzymatic complex that plays an essential role in many pathways of RNA processing and degradation. Here, we describe the structural characterization of the predicted archaeal exosome in solution using small angle x-ray scattering. The structure model calculated from the small angle x-ray scattering pattern provides an indication of the existence of a disk-shaped structure, corresponding to the "RNases PH ring" complex formed by the proteins aRrp41 and aRrp42. The RNases PH ring complex corresponds to the core of the exosome, binds RNA, and has phosphorolytic and polymerization activities. Three additional molecules of the RNA-binding protein aRrp4 are attached to the core as extended and flexible arms that may direct the substrates to the active sites of the exosome. In the presence of aRrp4, the activity of the core complex is enhanced, suggesting a regulatory role for this protein. The results shown here also indicate the participation of the exosome in RNA metabolism in Archaea, as was established in Eukarya.
Highlights
In Eukarya, the complex of 3Ј 3 5Ј exoribonucleases named exosome participates in the maturation of the small nuclear RNAs, small nucleolar RNAs, and rRNAs [1] as well as in the degradation of deadenylated mRNAs [2, 3] and aberrant pre-rRNAs [4]
In the present study we report the in vitro assembly of a protein complex corresponding to the archaeal exosome using recombinant proteins of Pyrococcus abyssi (PAB) and P. horikoshii (PH); archaeal rRNA processing protein 4, aRrp42 (PH1548), and aRrp41 (PH1549)
The ORFs corresponding to aRrp4, (PAB0419), aRrp41 (PH1549), and aRrp42 (PH1548) were amplified by PCR, cloned in E. coli expression vectors, and purified as described under “Experimental Procedures” (Fig. 1B)
Summary
The core components of the eukaryotic exosome assemble into a doughnut-shaped structure, with the six RNase PH-type proteins forming a hexameric ring, and three proteins, which contain S1 RNA binding domain, are located on top of the RNases PH ring. Analyses of these proteins separately or in complex by small angle x-ray scattering (SAXS) allowed us to propose a structural model for the archaeal exosome In this model aRrp and aRrp form a disk-shaped structure that correlates with the PNPase hexameric ring formed by the RNase PH domains, as predicted by sequence analysis (Fig. 1A). The functional implications of these data and its relationship with the eukaryotic exosome structure are discussed
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