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(A) shows the direct recognition of the promoter by the RNA polymerase, as occurs in bacteria. (B) shows recognition of the promoter by a DNA-binding protein which forms a platform onto which the RNA polymerase binds. This indirect mechanism occurs with eukaryotic and archaeal RNA polymerases.

Structures of exosome domains. Residues in red indicate phosphate binding regions and residues in yellow highlight RNA binding surfaces. Structures depicted in cartoons with helices as tubes and β-strands as arrows. A) RNase PH homodimerization interface (PDB ID = 1UDN). Phosphate binding residues include T125 and R126. RNA binding residues: R86, R92, R96 and R99. B) PNPase RNase PH 1/RNase PH 2 domain binding interface (PDB ID = 1E3P). Phosphate binding residues include: T462 and S463. RNA binding interface residues: R100, R104, R107, R422 and R423. A second RNA binding site includes residues F84, F85, R86 and R87. C) Archaeal S. solfataricus exosome Rrp41/Rrp42 heterodimer interface (PDB ID = 2JE6). Phosphate binding residues are from ssRrp41: S138 and R139. RNA binding interface residues from ssRrp41 are R98 and R99 and R112, R116 and R119 (ssRrp42). The second RNA binding region includes residues R67 and H68 (from ssRrp41) D) Eukaryotic H. sapiens Rrp41/Rrp45 hetero-dimerization interface. Putative RNA binding interface residues include: K94, S95, R104, R108 and R111 and the second putative RNA binding region includes residues R61 and A62. Structures for the archaeal three-component cap subunits (PDB ID = 2BAO and 2BA1): E) A. fulgidus Csl4 and F) A. fulgidus Rrp4. Putative RNA binding surfaces are highlighted in yellow for the S1 domain and KH domain on a transparent surface representation. Similar structures exist for the human three‑component cap subunits Csl4, Rrp4 and Rrp40 as discussed in the text (PDB ID = 2NN6).

Exemplary 4-thiouracil (4-TU) labeling of total RNA in S. cerevisiae and H. volcanii : (a) Workflow summary of 4-TU labeling. 4-thiouracil labeling and biotinylation workflow is briefly depicted. Growing cells (see Subheading 3.1.) are pulse labeled with 4-thiouracil as described in Subheading 3.2. After RNA extraction (see Subheading 3.3), sulfhydryl groups are specifically biotinylated in the presence of methylthiosulfonate activated Biotin derivate (MTS-Biotin) (see Subheading 3.4). After biotinylation RNA are separated by gel electrophoresis and visualized as described in Subheading 3.5 and below. (b and c) Exemplary 4-TU labeling applied on the eukaryotic model organism S. cerevisiae . The indicated yeast cells, wild-type (WT), cells depleted for the small and large ribosomal subunit biogenesis factors Pwp2 (GAL::PWP2) and Pol5 (GAL::POL5), respectively [39, 40] were labeled for 20 min with 4-thiouracil as indicated in Subheading 3.2. Labeled total RNA were separated by denaturing agarose (b) or polyacrylamide (c) gel electrophoresis, respectively. Biotinylated RNA was detected as described in Subheading 3.5. Ribosomal RNA precursors (35S, 27S, and 20S rRNA) and mature ribosomal RNA (25S, 18S, 5.8 and 5S rRNA) are indicated. Please note that depletion of the small ribosomal subunit biogenesis factor, Pwp2, for 14 h leads to the reduction of small ribosomal subunit pre- and mature rRNAs (20S and 18S rRNA, respectively) [39]. In contrast depletion of the large ribosomal subunit biogenesis factor, Pol5, for 9 h leads to the reduction of the common large ribosomal subunit precursor and corresponding mature rRNA (27S, 25S, and 5.8S, respectively) [39–41]. Additional application examples using this protocol and S. cerevisiae is described in [41]. (d) Exemplary 4-TU labeling applied on the archaeal model organism H. volcanii . Haloferax volcanii wildtype cells were labeled for 3 h with 4-thiouracil as indicated in Subheading 3.2. Labeled total RNA was separated by denaturing agarose electrophoresis. Biotinylated RNA was detected as described in Subheading 3.5. Mature ribosomal RNA (23S, 16S, and 5S rRNA) observed in these conditions are indicated. Additional application examples using this protocol and H. volcanii has been described previously [36]

'Exosomes' from bacteria, archaea and eukaryotes have a similar architecture. RNase PH, PNPase, archaeal exosome and eukaryotic core exosome structures and schematics are depicted in two orientations which we denote bottom and top. Architectures emphasize a six-component ring with or without phosphorolytic active sites (shown as red dots in cartoon representation and red surfaces in the surface representations of the respective structures). A) RNase PH. The Aquifex aeolicus RNase PH structure (PDB ID = 1UDN) forms a homohexamer of PH subunits (colored dark blue and light blue, for clarity). B) PNPase. The S. antibioticus PNPase structure (PDB ID = 1E3P) forms a homotrimer. PNPase protomers are colored light yellow, dark yellow and light brown to distinguish the homotrimer of RNase PH 1-like (PH 1) and RNase PH 2-like (PH 2) domains. The α domain was omitted to enable visualization of the phosphorolytic active sites in the bottom view (red dots). C) Archaeal exosome. The S. solfataricus archaeal exosome (PDB ID = 2JE6) is depicted with Rrp41 subunits (blue) and Rrp42 subunits (green) which form the six-component ring. Top view in the schematic shows the orientation of Csl4 (N-Terminal Domain, S1 domain and C-Terminal Domain) and Rrp4 (N-Terminal Domain, S1 domain and KH domain) labeled and shown in grey. The surface representation of the structure depicts the Rrp4-bound archaeal exosome. Residues in the active site are partially occluded from view such that the active site appears as two discontinuous red surfaces. D) Eukaryotic core exosome. The eukaryotic exosome is shown from H. sapiens (PDB ID = 2NN6). Subunits Rrp41 (magenta), Rrp42 (red), Mtr3 (orange), Rrp43 (yellow), Rrp46 (green), Rrp45 (blue) form the six-component ring. Subunits Rrp40 (light pink), Csl4 (cyan) and Rrp4 (grey) form the three-component cap. No phosphorolytic active site exists in the eukaryotic core exosome.