Role of stoichiometry between mRNA, translation factor SeIB and selenocysteyl-tRNA in selenoprotein synthesis

The specialized translation factor SeIB forms a quaternary complex in vitro with selenocysteyl-tRNA^Sec, the selenoprotein mRNA and guanine nucleotides. To gain information on whether this complex is required for selenocysteine insertion in vivo we have studied the effect of unbalanced ratios of the individual components of the complex on UGA readthrough. It was found that overproduction of SeIB in an otherwise wild-type genetic background reduced UGA read-through to less than 1 %. Concomitant overexpression of seIC (the gene for selenocysteine-specific tRNA^Sec) completely reversed the inhibition. Truncation of SeIB from the C-terminal end abolished function as a translation factor but the truncated molecules, when overproduced, were still able to suppress UGA read-through. The inhibition was also reversed by overproduction of tRNA^Sec. The most plausible explanation is that overproduction of SeIB impairs the statistics of formation of the quaternary complex and that the C-terminally truncated molecules are still able to bind selenocysteyl-tRNA^Sec and remove it from the pool. The mRNA-binding capacity, therefore, is physically separated from the selenocysteyl-tRNA-binding domain.