DOMAIN-STRUCTURE OF THE PROKARYOTIC SELENOCYSTEINE-SPECIFIC ELONGATION-FACTOR SELB

Incorporation of the non-canonical amino acid selenocysteine into prot eins requires the activity of the elongation factor SelB which substit utes for the function of EF-Tu. In contrast to EF-Tu, SelB binds selen ocystylated tRNA(Sec) and an mRNA secondary structure adjacent to the UGA selenocysteine codon. To gain information on the domain structure of this specialized translation factor, the selB genes from two bacter ia unrelated to Escherichia coli (Clostridium thermoaceticum and Desul fomicrobium baculatum) were cloned and sequenced. The derived amino ac id residue sequences were compared to those of SelB from E. coli and H aemophilus influenzae and to EF-Tu sequences. The alignment revealed t hat SelB contains all three domains characterized for EF-Tu. A fourth, C-terminally located domain shows only limited sequence conservation within the four SelB proteins. To elucidate the function of this C-ter minal part a structure-function analysis of SelB from E. coli was perf ormed. It showed that a C-terminal 17 kDa subdomain of the translation factor, when expressed separately, specifically binds the mRNA second ary structure. The recognition motif itself could be reduced to a 17 n ucleotide minihelix without loss of binding affinity and specificity. A truncated SelB lacking the mRNA binding domain was still able to int eract with selenocysteyl-tRNA(Sec). Expression of the mRNA binding dom ain alone suppressed selenocysteine insertion in vivo by competing wit h SelB for its binding site at the mRNA. The results indicate that Sel B can be considered as an EF-Tu homolog hooked to the mRNA via its C-t erminal domain. (C) 1996 Academic Press Limited