FUNCTIONALITY OF MUTATIONS AT CONSERVED NUCLEOTIDES IN EUKARYOTIC SECIS ELEMENTS IS DETERMINED BY THE IDENTITY OF A SINGLE NONCONSERVED NUCLEOTIDE

In eukaryotes, the specific cotranslational insertion of selenocystein e at UGA codons requires the presence of a secondary structural motif in the 3' untranslated region of the selenoprotein mRNA. This selenocy steine insertion sequence (SECIS) element is predicted to form a hairp in and contains three regions of sequence invariance that are thought to interact with a specific protein or proteins. Specificity of RNA-bi nding protein recognition of cognate RNAs is usually characterized by the ability of the protein to recognize and distinguish between a cons ensus binding site and sequences containing mutations to highly conser ved positions in the consensus sequence. Using a functional assay for the ability of wild-type and mutant SECIS elements to direct cotransla tional selenocysteine incorporation, we have investigated the relative contributions of individual invariant nucleotides to SECIS element fu nction. We report the novel finding that, for this consensus RNA motif , mutations at the invariant nucleotides are tolerated to different de grees in different elements, depending on the identity of a single non conserved nucleotide. Further, we demonstrate that the sequences adjac ent to the minimal element, although not required for function, can af fect function through their propensity to base pair. These findings sh ed light on the specific structure these conserved sequences may form within the element. This information is crucial to the design of strat egies for the identification of SECIS-binding proteins, and hence the elucidation of the mechanism of selenocysteine incorporation in eukary otes.