In eukaryotes, the specific cotranslational insertion
of selenocysteine at UGA codons requires the presence of
a secondary structural motif in the 3′ untranslated
region of the selenoprotein mRNA. This selenocysteine insertion
sequence (SECIS) element is predicted to form a hairpin
and contains three regions of sequence invariance that
are thought to interact with a specific protein or proteins.
Specificity of RNA-binding protein recognition of cognate
RNAs is usually characterized by the ability of the protein
to recognize and distinguish between a consensus binding
site and sequences containing mutations to highly conserved
positions in the consensus sequence. Using a functional
assay for the ability of wild-type and mutant SECIS elements
to direct cotranslational selenocysteine incorporation,
we have investigated the relative contributions of individual
invariant nucleotides to SECIS element function. We report
the novel finding that, for this consensus RNA motif, mutations
at the invariant nucleotides are tolerated to different
degrees in different elements, depending on the identity
of a single nonconserved nucleotide. Further, we demonstrate
that the sequences adjacent to the minimal element, although
not required for function, can affect function through
their propensity to base pair. These findings shed light
on the specific structure these conserved sequences may
form within the element. This information is crucial to
the design of strategies for the identification of SECIS-binding
proteins, and hence the elucidation of the mechanism of
selenocysteine incorporation in eukaryotes.