Characterization of the SBP2‐Dependent Selenocysteine Incorporation Complex

Selenocysteine incorporation involves a translational recoding event at specific UGA codons found upstream of stable stem‐loop structures known as SECIS elements. In addition, at least two trans‐acting factors are required: 1) a Sec‐specific elongation factor (eEFSec) and 2) a SECIS binding protein (SBP2). Truncations of SBP2 have revealed two distinct domains that include an RNA binding domain and a functional domain. Sedimentation through glycerol gradients has revealed that SBP2 is also stably associated with ribosomes, but the significance of this interaction in the context of Sec incorporation is unknown. Structurally, the SECIS element closely resembles the kink turn elements that are found in 28S rRNA, and we have previously hypothesized that SBP2 acts as a dimer to allow simultaneous interaction with a ribosomal kink‐turn and a SECIS element. However, a combination of gel filtration chromatography and co‐purification studies demonstrates that SBP2 does not self‐associate but is quantitatively bound to ribosomes and directly binds to kink turn elements found in 28S rRNA. Interestingly, a wild‐type but not mutant SECIS element is able to effectively compete with the SBP2/ribosome interaction, clearly indicating that SBP2 cannot simultaneously interact with the ribosome and the SECIS element. Presently, we are performing an alanine‐scanning mutagenesis of the RNA binding domain of SBP2 in order to precisely determine the residues required for ribosome binding and to establish the requirement for this interaction. To that end, we have developed a sucrose cushion assay to screen for ribosome binding defects. Based on these results we propose a revised model for Sec incorporation where SBP2 remains ribosome bound except during selenocysteine delivery to the ribosomal A‐site.