In vitro and in vivo characterization of novel mRNA motifs that bind special elongation factor SelB.
The special elongation factor SelB of Escherichia coli promotes selenocysteine incorporation into formate dehydrogenases. This is thought to be achieved through simultaneous binding to selenocysteyl-tRNASec and, in the case of formate dehydrogenase H, to an fdhF mRNA hairpin structure 3' adjacent to the UGA selenocysteine codon. By in vitro selection, novel RNA sequences ("aptamers"), which can interact tightly and specifically with SelB, were isolated from an RNA library. The library was comprised of mutagenized variants of the wild-type fdhF mRNA hairpin. One-half of the selected sequences contained the apical stem-loop of the fdhF mRNA hairpin highly conserved. Some of the aptamers showed deviations in the primary sequence within this region of the wild-type fdhF hairpin motif while still binding with high affinity to SelB. Binding studies performed with truncated versions of SelB revealed that aptamers binding to different sites on the protein have been selected. To dissect SelB binding to the fdhF hairpin from the overall biological 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 of selenocysteyl-tRNASec to the UGA codon.
Klug
S J
SJ
Institut für Biochemie der Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
Hüttenhofer
A
A
Kromayer
M
M
Famulok
M
M
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D017421:Sequence Analysis
D001426:Bacterial Proteins; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; C061878:SelB protein, Bacteria