Insight into mammalian selenocysteine insertion: Domain structure and ribosome binding properties of Sec insertion sequence binding protein 2

The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding o f a UGA stop codon as one specific for Sec. The recognition of UGA as Sec i n mammalian selenoproteins requires a Sec insertion sequence (SECIS) elemen t in the 3' untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using tru ncation and site-directed mutagenesis. We have localized the RNA binding do main to a conserved region shared with several ribosomal proteins and eukar yotic translation termination release factor 1. We also identified a separa te and novel functional domain N-terminal to the RNA binding domain which w as required for Sec insertion but not for SECIS binding. Conversely, we sho wed that the RNA binding domain was necessary but not sufficient for Sec in sertion and that the conserved glycine residue within this domain was requi red for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal. fraction of cell lysates an d that this interaction was not dependent on its SECIS binding activity, Th is interaction also occurred with purified components in vitro, and we pres ent data which suggest that the SBP2-ribosome interaction occurs via 28S rR NA SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.