In vitro selection of RNA aptamers that bind special elongation factor SelB, a protein with multiple RNA-binding sites, reveals one major interaction domain at the carboxyl terminus.
The SelB protein of Escherichia coli is a special elongation factor required for the cotranslational incorporation of the uncommon amino acid selenocysteine into proteins such as formiate dehydrogenases. To do this, SelB binds simultaneously to selenocysteyl-tRNA(Sec) and to an RNA hairpin structure in the mRNA of formiate dehydrogenases located directly 3' of the selenocysteine opal (UGA) codon. The protein is also thought to contain binding sites allowing its interaction with ribosomal proteins and/or rRNA. SelB thus includes specific binding sites for a variety of different RNA molecules. We used an in vitro selection approach with a pool completely randomized at 40 nt to isolate new high-affinity SelB-binding RNA motifs. Our main objective was to investigate which of the various RNA-binding domains in SelB would turn out to be prime targets for aptamer interaction. The resulting sequences were compared with those from a previous SELEX experiment using a degenerate pool of the wild-type formiate dehydrogenase H (fdhF) hairpin sequence (Klug SJ et al., 1997, Proc. Natl. Acad. Sci. USA 94:6676-6681). In four selection cycles an enriched pool of tight SelB-binding aptamers was obtained; sequencing revealed that all aptamers were different in their primary sequence and most bore no recognizable consensus to known RNA motifs. Domain mapping for SelB-binding aptamers showed that despite the different RNA-binding sites in the protein, the vast majority of aptamers bound to the ultimate C-terminus of SelB, the domain responsible for mRNA hairpin binding.
Klug
S J
SJ
Institut für Biochemie der Universität München, Germany.
Hüttenhofer
A
A
Famulok
M
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015723:Gene Library; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D017423:Sequence Analysis, RNA; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D012335:RNA, Ribosomal; C061878:SelB protein, Bacteria; D012313:RNA; D004247:DNA; D012343:RNA, Transfer; D012321:DNA-Directed RNA Polymerases; D010444:Peptide Elongation Factor Tu