RNA recognition by designed peptide fusion creates "artificial" tRNA synthetase.
The genetic code was established through aminoacylations of RNA substrates that emerged as tRNAs. The 20 aminoacyl-tRNA synthetases (one for each amino acid) are ancient proteins, the active-site domain of which catalyzes formation of an aminoacyl adenylate that subsequently reacts with the 3' end of bound tRNA. Binding of tRNA depends on idiosyncratic (to the particular synthetase) domains and motifs that are fused to or inserted into the conserved active-site domain. Here we take the domain for synthesis of alanyl adenylate and fuse it to "artificial" peptide sequences (28 aa) that were shown previously to bind to the acceptor arm of tRNAAla. Certain fusions confer aminoacylation activity on tRNAAla and on hairpin microhelices modeled after its acceptor stem. Aminoacylation was sensitive to the presence of a specific G:U base pair known to be a major determinant of tRNAAla identity. Aminoacylation efficiency and specificity also depended on the specific peptide sequence. The results demonstrate that barriers to RNA-specific aminoacylations are low and can be achieved by relatively simple peptide fusions. They also suggest a paradigm for rationally designed specific aminoacylations based on peptide fusions.
Frugier
Magali
M
Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, Unité Propre de Recherche 9002, Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, F-67084 Strasbourg Cedex, France.
Giege
Richard
R
Schimmel
Paul
P
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005821:Genetic Techniques; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
D010455:Peptides; D011993:Recombinant Fusion Proteins; D012313:RNA; D000604:Amino Acyl-tRNA Synthetases