1. Volume 55, Issue 6, Pages 856-867 (September 2014)
The Molecular Architecture of the TRAMP Complex Reveals the Organization and Interplay of Its Two Catalytic Activities 
Sebastian Falk, John R. Weir, Jendrik Hentschel, Peter Reichelt, Fabien Bonneau, Elena Conti 
Molecular Cell 
Volume 55, Issue 6, Pages (September 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 55, 856-867DOI: (10.1016/j.molcel.2014.07.020)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
The DExH Core of Mtr4 Interacts with Low-Complexity Regions of Trf4 and Air2
(A) Schematic domain organization of TRAMP subunits. Lines indicate low-complexity regions, and arrows indicate the regions previously crystallized (Hamill et al., 2010; Jackson et al., 2010; Weir et al., 2010).
(B) ITC experiments of Trf Air with Mtr4-Δ80 (left panel) and Mtr4-Δ80ΔSK (right panel). The calorimeter cell was filled with Trf4-Air2 at 10 μM, and Mtr4 was injected at 100 μM concentration consecutively in 10 μl volumes. In each inset are the number of calculated binding sites (N) and association and dissociation constants (Ka and Kd), calculated with the program Origin.
(C) ITC experiments of Mtr4-Δ80 with Trf Air (left panel) and Trf Air (right panel), as described in (B).
(D) Protein coprecipitations by nickel or GST pull-down assays. GST-His-Trf and either GST-His Air21-62 or Ztag-His-Air21-62 were incubated with Mtr4-Δ80ΔSK in a buffer containing 150 mM NaCl before coprecipitation with nickel-Sepharose (N) or GSH-Sepharose beads (G), as indicated. A total of 5% of the input (left lanes) and 10% of the eluates (right lanes) were analyzed on Coomassie-stained 4%–12% gradient gel SDS-PAGE (Nupage, Invitrogen).
(E) The N termini of MDM2 and MDMX (indicated in the structure on the right) were linked with Trf and Air21-62 to form an artificial heterodimer. The central panel shows a size-exclusion chromatography profile of Mtr4-Δ80ΔSK in the absence and presence of the Trf MDM2 - Air21-62-MDMX heterodimer. A Coomassie-stained 15% SDS PAGE gel with the corresponding peak fractions is on the right.
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4. Figure 2
Crystal Structure of Mtr4 Bound to Low-Complexity Regions of Trf4 and Air2
(A) The crystal structure of the S. cerevisiae Mtr4-Δ80-Trf4N-Air2N complex is shown in two orientations related by a 60° rotation around the horizontal axis. The Mtr4 DExH-helicase core is shown in yellow and the stalk/KOW insertion domain in green. Air2N is colored blue and Trf4N pink. Residue numbers of Air2N and Trf4N are indicated.
(B) Zoom-in view of the Trf4N interaction interface with the RecA2 domain of Mtr4, shown in a similar orientation as in Figure 2A, left panel (front view). Conserved residues involved in the interaction are shown as sticks.
(C and D) Zoom-in views of the Air2N interaction interfaces with the helical bundle and RecA2 domains of Mtr4, shown in a similar orientation as in Figure 2A, right panel. Conserved residues involved in the interaction are shown as sticks.
(E) Sequence alignment of the Mtr4-binding regions of S. cerevisiae (Sc) Trf4 and Trf5 and S. pombe (Sp) Cid14 (upper panel), and of Sc Air2 and Air1 and Sp Air1 (lower panel). Above the sequence, the residues of Trf4N or AirN that interact with the DExH-helicase core of Mtr4 are labeled with a yellow circle. The residues of Air2N that are involved in crystal contacts and bind to the KOW-domain of Mtr4 are highlighted with green triangles.
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5. Figure 3
Trf4-Air2 and Trf5-Air1 Use the Same Interaction Surface on Mtr4
(A) Recombinant wild-type and mutants of Mtr4-Δ80ΔSK were purified to homogeneity and tested in GST pull-down assays with GST-His -Trf and GST-His-Air21-62 (as described in Figure 1D) (lanes 2–5). In lanes 6–9 the corresponding pull-down assays with GST-His-Trf and GST-His-Air11-75 are shown.
(B) Recombinant wild-type and mutants of GST-His-Trf or GST-His-Air21-62 were purified to homogeneity and tested in GST pull-down assays with Mtr4-Δ80ΔSK (as described in Figure 1D).
(C) Growth assay of wild-type and mutant mtr4 strains. Endogenous MTR4 was replaced with wild-type or mutant mtr4-EGFP fusions. Cells were grown to early exponential phase, and serial dilutions were spotted onto 5-fluoroorotic acid (FOA) medium or control plates. Medium containing FOA selects for the loss of the rescue vector. SC, synthetic complete medium; YPAD, yeast extract peptone adenine dextrose; FOA, 5-fluoroorotic acid; Ura, uracil.
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6. Figure 4 SAXS Reconstructions of the TRAMP Complex
(A) Distance distribution functions P(r) from small-angle X-ray scattering data of Mtr4-Δ80-Trf Air (TRAMP-Δ80, in green) and Mtr4-Δ80ΔSK-Trf Air (TRAMP-Δ80ΔSK, in black). The SAXS data are in Figure S4A. The P(r) functions were generated with GNOM/AutoGNOM and normalized to unity at their maxima.
(B) Overall parameters computed directly from the scattering data of the different samples: the radius of gyration (Rg), the maximum particle dimension (Dmax), and the correlation coefficient (CC) (see Supplemental Experimental Procedures for details).
(C) The average SAXS envelope of the TRAMP-Δ80 complex (left panel) and the TRAMP-Δ80ΔSK complex (right panel) were calculated with GASBOR (Svergun et al., 2001) and displayed in transparent, volumetric representations. The Mtr4-Δ80 - Trf Air structure and the Trf Air structure (Hamill et al., 2010) were docked into the SAXS envelope as described in the text. The atomic models are colored according to the schemes in Figure 1A (DExH core in yellow, insertion domain in green, Air2 in blue, and Trf4 in pink). The unaccounted density likely corresponds to the regions of Trf4-Air2 not present in the crystal structures.
(D) Mapping of lysines protected in the TRAMP complex from crosslinking on the Mtr4-Δ80 - Trf4N - Air2N structure. The structure of the complex is shown in two orientations related by a 90° rotation around the horizontal axis. Mtr4-Δ80 is colored gray, Trf4N pink, and Air2N blue. In red are lysine residues protected in the TRAMP-Δ80ΔSK complex that were identified by mass spectrometry analysis of a crosslinked sample.
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7. Figure 5 Interplay of the Two ATPase Activities in the TRAMP Complex
(A) The Coomassie-stained 4%–12% gradient gel SDS-PAGE (Nupage, Invitrogen) shows the wild-type and mutant TRAMP-Δ80 complexes used in the assays in (B). The mutants lack either Trf4 polyadenylation activity (p− mutants containing Trf4-DADA) or Mtr4 ATPase activity (h− mutants, containing Mtr4-Δ80 K177A).
(B) The top panel shows a poly(A)polymerase assay. A 5′ 32P-labeled tRNAiMet (250 nM) was incubated at 20°C for the amount of time indicated with 500 nM of purified TRAMP complexes (in A) and 0.5 mM ATP using optimal buffer conditions (see the Experimental Procedures). The samples were separated on 10% denaturing gels. The lower panel shows a thin-layer chromatography analysis of Mtr4 and Trf4 activities in the TRAMP complex. The assay was performed in the same conditions, except that the RNA substrate was not labeled and ATP was spiked with α32P ATP. Samples were separated on polyethyleneimine-cellulose.
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8. Figure 6 Model for the Mechanism of the TRAMP Complex
The schematic representation of the TRAMP complex recapitulates the structural and biophysical results from previous studies (Hamill et al., 2010; Jackson et al., 2010; Weir et al., 2010) and from this work, and shows a possible mode of action for unmodified tRNAiMet degradation.
(A) The DExH helicase core of Mtr4 is shown in yellow (with the internal RNA channel highlighted), and the insertion domain in green Trf4 is shown in pink with the polyadenylation active site indicated. Air2 is in blue. The N-terminal domains of Trf4 and Air2 interact with the circumference of the DExH core. Air2N latches onto the KOW domain.
(B) tRNA is recognized by TRAMP, possibly by the insertion domain of Mtr4 (Hamill et al., 2010; Jackson et al., 2010; Weir et al., 2010) and the ZK1-3 of Air2 (Hamill et al., 2010).
(C and D) (C) The tRNA 3′ end is threaded through the helicase channel and (D) reaches the polyadenylation site of Trf4.
(E) As the activity of Trf4 is distributive (LaCava et al., 2005), detachment of the RNA from the polyadenylation site would allow the RNA 3′ end to be accessible to the exosome. It is possible that different RNA substrates might be directly degraded after emerging from the helicase core (bypassing polyadenylation) or are directly polyadenlyated (bypassing unwinding), rationalizing why the ATPase and polyadenylation activities of TRAMP are essential in some cases (Vanácová et al., 2005), but not in others (Callahan and Butler, 2008).
Molecular Cell  , DOI: ( /j.molcel )
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