Characterization of the yeast CE and RE complexes. (a) SDS/PAGE analysis of the CE (lane 1) and RE (lanes 2 and 3) complexes. The Csl4 protein is near stoichiometric in lane 3. Identity of each exosome subunit, including the two proteolyzed Rrp44 fragments (marked with *) in lanes 2 and 3, was confirmed by using mass spectrometry. (b) Gel-filtration profile showing that RE migrates as a bigger complex than CE. (c and d) Negative-stain EM of CE (c) and RE (d) complexes. (Scale bars: 50 nm.)

Single-particle reconstruction of the yeast CE and RE complexes. (a) Front, back, and top views of the yeast RE reconstruction. Note that the Fourier shell correlation (FSC) analysis (graph) indicates that the RE reconstruction is at 19-Å resolution by 0.5 criterion. (b) Three views of the CE reconstruction (in the same orientations as the RE complex). The Fourier shell correlation curve (graph) indicates 23-Å resolution for the yeast CE reconstruction. For comparison, the human CE crystal structure was low-pass-filtered to 23-Å resolution and shown in the same views as Insets (scaled at 35% of the yeast CE maps). The arrowhead points to the Csl4 density, which is absent in the yeast CE reconstruction. This result further demonstrates that the projection-matching reconstruction did not introduce model bias. (c) Difference map between the CE and RE (mesh) reconstructions after the alignment of the two. Densities that are present in the RE, but absent in the CE, reconstruction are shown in gold, whereas the densities present in the CE, but not in the RE, reconstruction are shown in cyan. Densities corresponding to the core and Rrp44 protein are indicated, and the head and body region assignment of Rrp44 is marked.

Docking of atomic models into the EM reconstructions. Crystal structures of the human CE and the E. coli RNase II were docked into the yeast RE EM envelope. See text for docking procedure details. (a) Front, back, and top views of the RE EM envelope with the docked CE and RNase II models. The central channels inside the core and Rrp44 protein are marked with dashed arrows. (b) A slice at the CE region perpendicular to the central solvent-accessible channel. The structures of the eight CE subunits are colored the same as their labels. (c) The EM envelope of the Rrp44 protein with the docked E. coli RNase II model at the Rrp44 body region. Landmarks on the RNase II (the CSD, RNB, and S1 domains) are labeled with the same color as their ribbon diagram. The entry and exit of RNA substrate of RNase II are indicated by arrows. Helices 9–11 of RNase II, which protrude partially from the density map, are indicated by arrowheads. Note the 20-Å gap between the head and body, which we believe restricts the access of the RNA substrates.

Sequence alignment for the Rrp44 N-terminal region. Rrp44 sequences from budding yeast, fission yeast (Schizosaccharomyces pombe, SCHPO), human, mouse, Xenopus laevis, and Drosophila melanogaster were aligned by using ClustalW (red for identical residues, yellow for similar ones). The secondary structure predictions also are shown. Regions corresponding to the PINc domain (amino acids 86–207) and two OB folds (amino acids 277–366 and 393–464, equivalent to the two CSDs in E. coli RNase II) are highlighted in red and blue closed bars, respectively.

Conservation analysis of the RE interacting interface. The RE reconstruction is sliced at the interface, and the Rrp44 and the CE were rotated 90° to reveal the interaction surface. Based on the multiple sequence alignment of the exosome subunits by using ClustalW, a conservation map for the bottom of the CE was rendered by using the Multialign viewer function in Chimera with the conservative histogram calculated by using program AL2CO (42). The correlated interacting surfaces on the core and Rrp44 are marked with circles of the same color. The conservation map of the Rrp42 surface, which does not contact Rrp44, is shown for comparison.

Possible RNA processing mechanism by the yeast RE complex. (a) In the absence of the CE, the head of Rrp44 can tumble freely relative to the body and does not interfere with the enzymatic activity in the body region. The CE adopts a rigid conformation regardless of Rrp44's presence. (b) Binding of Rrp44 to the CE positions its head 20 Å away from its body, resulting in down-regulation of Rrp44's activity due to a steric hindrance effect in substrate recruitment. (c) RNA substrates can access the Rrp44 body domain active site either through the central channel of the CE (through exosome route) or directly from the ventral side of Rrp44 (direct access route). The two routes are not mutually exclusive.