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

The specialized translation factor SelB 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 SelB in an otherwise wild-type genetic background reduced UGA readthrough to less than 1%. Concomitant overexpression of selC (the gene for selenocysteine-specific tRNA(Sec)) completely reversed the inhibition. Truncation of SelB 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 SelB 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.