IN-VITRO AND IN-VIVO CHARACTERIZATION OF NOVEL MESSENGER-RNA MOTIFS THAT BIND SPECIAL ELONGATION-FACTOR SELB

The special elongation factor SelB of Escherichia coli promotes seleno cysteine incorporation into formate dehydrogenases, This is thought to be achieved through simultaneous binding to selenocysteyl-tRNA(Sec) a nd, in the case of formate dehydrogenase H, to an fdhF mRNA hairpin st ructure 3' adjacent to the UGA selenocysteine codon, By in vitro selec tion, novel RNA sequences (''aptamers''), which can interact tightly a nd specifically with SelB, were isolated from an RNA library, The libr ary was comprised of mutagenized variants of the wild-type fdhF mRNA h airpin, One-half of the selected sequences contained the apical stem-l oop of the fdhF mRNA hairpin highly conserved, Some of the aptamers sh owed deviations in the primary sequence within this region of the wild -type fdhF hairpin motif while still binding with high affinity to Sel B, Binding studies performed with truncated versions of SelB revealed that aptamers binding to different sites on the protein have been sele cted, To dissect SelB binding to the fdhF hairpin from the overall bio logical function of this complex, four selected aptamers were analyzed in vivo for UGA readthrough in a lacZ fusion construct, Among these, one promoted UGA readthrough in vivo. Three of the aptamers, however, were drastically reduced or unable to replace the fdhF mRNA hairpin in vivo, despite the similar secondary structure and binding affinities of these RNAs compared with the wild-type motif, This finding implies functions of the fdhF hairpin that go beyond the mere tethering off se lenocysteyl-tRNA(Sec) to the UGA codon.