A Conserved Domain within Arc1p Delivers tRNA to Aminoacyl-tRNA Synthetases  George Simos, Anke Sauer, Franco Fasiolo, Eduard C Hurt  Molecular Cell  Volume 1, Issue 2, Pages 235-242 (January 1998) DOI: 10.1016/S1097-2765(00)80024-6

Figure 1 The N Domain of Arc1p Binds to MetRS and GluRS, while the M and C Domains of Arc1p Are Required for tRNA Binding (A) Schematic representation of the Arc1p deletion mutants used in this study. The N-terminal (N), middle (M), and C-terminal (C) domains are indicated. Numbers refer to amino acid residues. (B) Total cell extracts containing ProtA-Arc1p deletion mutants were passed through an IgG-Sepharose affinity chromatography column and bound proteins were eluted and analyzed by SDS–PAGE and Coomassie staining (upper panel) or probed with an anti-MetRS antibody (lower panel). (C) The deletion mutants of Arc1p, expressed and purified from E. coli, were analyzed by SDS–PAGE and Coomassie staining. (D) Each recombinant Arc1p form was tested for binding to in vitro transcribed and radiolabeled (P32) yeast initiator tRNAMet (120 nM) using a band shift assay. Lane 1, no protein; lane 2, full-length Arc1p (0.25 μg); lane 3, Arc1-ΔN (0.25 μg); lanes 4–7, N domain (0.25, 1, 2.5, and 5 μg, respectively); lanes 8–11, C domain (0.25, 1, 2.5, and 5 μg); lanes 12–15, Arc1-ΔM (0.25, 1, 2.5, and 5 μg); and lanes 16–19, Arc1-ΔC (0.25, 1, 2.5, and 5 μg). Molecular Cell 1998 1, 235-242DOI: (10.1016/S1097-2765(00)80024-6)

Figure 2 Cooperative and Specific Binding of the Arc1p/MetRS Complex to tRNA (A) Recombinant Arc1p deletion forms (0.2 μM) were tested for binding to in vitro transcribed and radiolabeled (P32) yeast initiator tRNAMet (40 nM) using a band shift assay in the absence (lanes 1–5) or in the presence of equimolar amounts of MetRS (lanes 6–10). A supershift is observed in the case of Arc1p (compare lanes 2 and 7), Arc1-ΔC (lanes 4 and 9), and Arc1-ΔM (lanes 5 and 10). (B) The Arc1-ΔM (left panel) and the Arc1-ΔC (right panel) forms were tested for binding to radiolabeled yeast initiator tRNAMet in the presence of MetRS. Binding was competed with a 50-fold excess of unlabeled RNA molecules as indicated. (C) Binding of Arc1p (0.1 μM) to tRNAMet (40 nM) in the absence (top panel) or in the presence (bottom panel) of MetRS (0.1 μM) was competed with increasing amounts of the cognate or a noncognate tRNA as indicated (10- to 50-fold excess). Molecular Cell 1998 1, 235-242DOI: (10.1016/S1097-2765(00)80024-6)

Figure 3 High Affinity Association of tRNA with Cognate Aminoacyl-tRNA Synthetases Is Mediated In Vivo by the Arc1p tRNA-Binding Domain (A) RNA from yeast lysates (lanes 1–6) or RNA associated with the MetRS/GluRS/Arc1 complex bound on the IgG-Sepharose beads through the protein A tag of Arc1p (lanes 7–12, refer also to Figure 1B) was extracted and analyzed by denaturing polyacrylamide gel electrophoresis and ethidium bromide staining (upper panel), or Northern blot using specific probes for the indicated tRNAs. Two tRNA species corresponding to elongator tRNAMet and tRNAGlu were found to specifically associate with the complex containing wild-type Arc1p. tRNAGlu could also be detected after long exposure when the M or the C domain or both were missing from Arc1p, but in drastically lower amounts. (B) RNA was extracted from total yeast extracts containing ProtA-Arc1p (lane 1), the flow-through of the IgG-Sepharose column (lane 2) and the MetRS/GluRS/Arc1p/tRNAMet/tRNAGlu complex that bound to the column after incubation in the presence (lane 3) or absence (lane 4) of methionine and ATP. The integrity of the extracted RNA was confirmed by denaturing polyacrylamide gel electrophoresis and ethidium bromide staining (upper panel). The extent of aminoacylation of tRNAMet is shown by the mobility shift in an acid-urea polyacrylamide gel detected by Northern blot (bottom panel). Molecular Cell 1998 1, 235-242DOI: (10.1016/S1097-2765(00)80024-6)

Figure 4 Specific Genetic Interaction of Arc1p Domains with MetRS, GluRS, Los1p, and Nsp1p Five arc1 synthetic lethal (sl) mutants were transformed with the various mutant forms of Arc1p, and complementation is shown by the ability to lose the pURA3-ARC1 plasmid and grow in the presence of 5-FOA. (A) The sl mutant slk117 that carries a mutation in the MetRS gene and lacks Arc1p is not viable unless transformed by plasmids expressing proteins that contain the N-terminal domain of Arc1p. (B) The sl mutant slk88 that carries a mutant GluRS gene and lacks Arc1p is not viable unless transformed by plasmids expressing the ΔC or ΔM forms of Arc1p. (C and D) The sl mutant sl29 that carries a mutation in the ARC1 gene and lacks Los1p (C) and the sl mutant sl276 that has a mutant NSP1 gene and lacks Arc1p (D) are not viable unless transformed by plasmids expressing proteins that contain the C-terminal domain of Arc1p. (E and F) The double disruption mutant that lacks LOS1 and ARC1 is not viable, unless transformed with a plasmid expressing the Arc1-ΔM form that contains both the N-terminal and C-terminal domains (E). The same mutant can grow even when these domains are expressed from different plasmids as separate polypeptides (F). Molecular Cell 1998 1, 235-242DOI: (10.1016/S1097-2765(00)80024-6)

Figure 5 A Model for the Function of Arc1p (A) Schematic alignment of Arc1p with E. coli, yeast, and C. elegans MetRS; the β subunit of E. coli PheRS; and human TyrRS. The hatched boxes represent the TRBD domain (hatched lines indicate the lysine-rich area that in the case of Arc1p corresponds to the M domain). The lightly shaded box indicates the conserved MetRS part that contains the catalytic core. The appended N-terminal domain in the yeast MetRS is represented by the darkly shaded box. (B) A hypothetical model for the interaction of MetRS with tRNA when the TRBD (hatched pattern) is fused to the enzyme (in E. coli and C. elegans, left) or when it is part of Arc1p (in yeast and human, right). The model on the left may also apply to E. coli PheRS and human TyrRS, while the one on the right to yeast and human GluRS. Molecular Cell 1998 1, 235-242DOI: (10.1016/S1097-2765(00)80024-6)