Volume 11, Issue 3, Pages 773-781 (March 2003) Coupled tRNASec-Dependent Assembly of the Selenocysteine Decoding Apparatus  Ann Marie Zavacki, John B. Mansell, Mirra Chung, Boris Klimovitsky, John W. Harney, Marla J. Berry  Molecular Cell  Volume 11, Issue 3, Pages 773-781 (March 2003) DOI: 10.1016/S1097-2765(03)00064-9 Copyright © 2003 Cell Press Terms and Conditions

Figure 1 The C-Terminal Domain of EFsec Coimmunoprecipitates In Vitro with SBP2, but the Full-Length Protein Does Not Constructs expressing the indicated amino acids from EFsec were transcribed and translated in vitro in rabbit reticulocyte lysates. In vitro translated c-myc-tagged SBP2 was added to reactions, and coimmunoprecipitations were performed with anti-c-myc-agarose, followed by SDS-PAGE and autoradiography. Lanes 1–4, in vitro translation reactions; lanes 5–8, coimmunoprecipitations. Molecular Cell 2003 11, 773-781DOI: (10.1016/S1097-2765(03)00064-9) Copyright © 2003 Cell Press Terms and Conditions

Figure 2 EFsec Constructs Used for Interaction Domain Mapping Constructs expressing the indicated regions of EFsec were subcloned into pGBK with N-terminal HA epitope tags. Black lines indicate exon-exon boundaries. Molecular Cell 2003 11, 773-781DOI: (10.1016/S1097-2765(03)00064-9) Copyright © 2003 Cell Press Terms and Conditions

Figure 3 Discrete Regions in the C-Terminal Domain of EFsec Interact with SBP2 Constructs expressing the indicated amino acids from EFsec were transcribed and translated in vitro in rabbit reticulocyte lysates or were expressed as GST-fusions in E. coli. C-myc-SBP2 was translated in vitro, or GST-SBP2 was expressed in E. coli, as described in Experimental Procedures. Coprecipitations were performed with anti-c-myc-agarose (A) or glutathione-agarose (B and C), followed by SDS-PAGE and autoradiography. Left panels, in vitro translation reactions; right panels, coprecipitations or GST pull-downs. Molecular Cell 2003 11, 773-781DOI: (10.1016/S1097-2765(03)00064-9) Copyright © 2003 Cell Press Terms and Conditions

Figure 4 Role of EFsec C-Terminal Domain Conserved Amino Acids in EFsec-SBP2 Interaction (A) Alignment of EFsec C-terminal domain sequences. Conserved amino acids are highlighted in gray, and mutated amino acids are indicated by asterisks. (B) GST pull-downs of EFsec C-terminal domain mutants. GST-fusion constructs expressing the EFsec 448–583 C-terminal domain with the indicated mutations were expressed in E. coli, and crude extracts were incubated with in vitro translated SBP2, followed by pull-down with glutathione-agarose, SDS-PAGE, and autoradiography (upper panel). Aliquots of bacterially expressed EFsec mutants were analyzed by Western blotting with an anti-GST antibody to verify expression levels (lower panel). Molecular Cell 2003 11, 773-781DOI: (10.1016/S1097-2765(03)00064-9) Copyright © 2003 Cell Press Terms and Conditions

Figure 5 Effect of tRNASec and SBP2 Expression on EFsec-SBP2 Coimmunoprecipitation and EFsec Protein Levels In Vivo (A) EFsec full-length protein (full) or 448–583 C-terminal domain (C term) with N-terminal FLAG epitope tags was coexpressed in transfected HEK-293 cells with SBP2 in the presence or absence of the tRNASec gene. Cell lysates were subjected to immunoprecipitation with anti-SBP2 antibody (upper panel). Aliquots were analyzed by Western blotting with anti-FLAG antibody (lower panel). (B) FLAG-tagged EFsec full-length protein or 448–583 C-terminal domain was expressed as above in the presence or absence of SBP2 and tRNASec expression plasmids, as indicated. Cell lysates were analyzed by Western blotting with anti-FLAG antibody. (C and D) 35S-methionine in vivo labeling was carried out following expression of the indicated plasmids in transfected cells. After 1 hr of labeling, media were changed and unlabeled methionine was added. Incorporation of unlabeled methionine and decay of labeled proteins was allowed to proceed for the indicated times, followed by immunoprecipitation with anti-FLAG antibody, SDS-PAGE, and autoradiography. Molecular Cell 2003 11, 773-781DOI: (10.1016/S1097-2765(03)00064-9) Copyright © 2003 Cell Press Terms and Conditions

Figure 6 The C-Terminal Domain of EFsec Competes for Selenocysteine Incorporation In Vivo EFsec 448–583 C-terminal fragment was expressed in transfected HEK-293 cells with a type 1 deiodinase selenoenzyme expression plasmid or a cysteine mutant type 1 deiodinase. Type 1 deiodinase enzyme activity was assayed in cell lysates as described previously. Molecular Cell 2003 11, 773-781DOI: (10.1016/S1097-2765(03)00064-9) Copyright © 2003 Cell Press Terms and Conditions

Figure 7 Model for the Effects of tRNA Binding on EFsec Conformation, EFsec-SBP2 Interaction, and Ribosome Interaction (A) Binding of tRNASec by EFsec triggers a conformational change, indicated by bending of the C-terminal domain upward, allowing interaction with SBP2. SBP2 can then deliver tRNASec to the UGA codon at the ribosome. (B) Delivery of Sec-tRNA at the ribosome triggers a reversion of EFsec to the pre-tRNA binding conformation, which disfavors SBP2 interaction, releasing EFsec to bind a new Sec-tRNASec molecule and allowing SBP2 to recruit a new Sec-tRNASec-EFsec complex. The mRNA, including the SECIS element in the 3′UTR, is indicated by the thin black line. The open reading frame is indicated by the thick black line, with double gray ovals representing ribosomes on the mRNA. SBP2 and EFsec are labeled, with the C-terminal domain of EFsec depicted by the dark gray half-oval. Molecular Cell 2003 11, 773-781DOI: (10.1016/S1097-2765(03)00064-9) Copyright © 2003 Cell Press Terms and Conditions