Dissescting the role of SBP2 in selenocysteine incorporation

Abstract

The SECIS binding protein, SBP2, is required for the co‐translational incorporation of selenocysteine (Sec) at specific UGA codons. The C‐terminal 447 residues of SBP2 (CTSBP2) are sufficient to promote Sec incorporation and have been divided into a functional domain (FxD) and an RNA binding domain (RBD). SECIS and ribosome interactions through the RBD are well characterized but it is not known how the FxD participates in Sec incorporation. To determine the role of the FxD in Sec incorporation we created chimeras using the domains of CTSBP2 and the analogous region of SBP2‐like protein (SLP) to determine if the FxD in SLP has sufficient identity with SBP2 to promote Sec incorporation. Using an in vitro luciferase reporter specific for Sec incorporation, we showed that the FxD in SLP cannot promote Sec incorporation. A homology driven approach was used to mutate FxD residues in groups of five and mutant proteins were assayed for their ability to promote Sec incorporation, SECIS binding, and ribosome binding. We identified three regions critical for Sec incorporation that implicate the FxD in ribosome binding and suggest that SBP2's ribosome interaction is dynamic. Additionally, we expressed the FxD and RBD as separate recombinant proteins and assayed them for their ability to promote Sec incorporation. Interestingly, we found that both domains together in a Sec incorporation assay are competent but gel filtration and EMSA studies failed to show a stable interaction of the two domains. We currently hypothesize that the FxD acts to allow Sec‐tRNASec access to the ribosomal A‐site and are developing assays to test this.This work was supported by PHS grants T32AI007403 (JD), FGM081920A (KC), and GM077073 (PRC).