The amino acid selenocysteine is encoded by UGA, usually a stop codon, thus 
requiring a specialized machinery to enable its incorporation into 
selenoproteins. The machinery comprises the tRNA(Sec), a 3'-UTR mRNA stem-loop 
termed SElenoCysteine Insertion Sequence (SECIS), which is mandatory for 
recoding UGA as a Sec codon, the SECIS Binding Protein 2 (SBP2), and other 
proteins. Little is known about the molecular mechanism and, in particular, 
when, where, and how the SECIS and SBP2 contact the ribosome. Previous work by 
others used the isolated SECIS RNA to address this question. Here, we developed 
a novel approach using instead engineered minimal selenoprotein mRNAs containing 
SECIS elements derivatized with photoreactive groups. By cross-linking 
experiments in rabbit reticulocyte lysate, new information could be gained about 
the SBP2 and SECIS contacts with components of the translation machinery at 
various translation steps. In particular, we found that SBP2 was bound only to 
the SECIS in 48S pre-initiation and 80S pretranslocation complexes. In the 
complex where the Sec-tRNA(Sec) was accommodated to the A site but 
transpeptidation was blocked, SBP2 bound the ribosome and possibly the SECIS 
element as well, and the SECIS had flexible contacts with the 60S ribosomal 
subunit involving several ribosomal proteins. Altogether, our findings led to 
broadening our understanding about the unique mechanism of selenocysteine 
incorporation in mammals.