Isolated RNA binding domain of a class I tRNA synthetase.
Class I tRNA synthetases typically have two major domains consisting of a class-defining N-terminal nucleotide binding fold, which contains the active site, and an idiosyncratic C-terminal domain, which in many instances provides for interactions with the tRNA anticodon. Whether the C-terminal domain can function in specific RNA binding when disconnected from the catalytic core is not known. We fused the anticodon binding domain of Escherichia coli methionyl tRNA synthetase to maltose binding protein. This fusion protein and the released, isolated domain are stable and have native-like structural characteristics, as shown by circular dichroism and thermal denaturation studies. Both the fusion protein and the isolated domain bind specifically to a small RNA hairpin oligonucleotide that recapitulates the anticodon stem-loop of tRNAfMet. Neither protein binds to an RNA hairpin with a point mutation in the anticodon trinucleotide. The binding specificity and affinity of these proteins duplicate those of the interaction between methionyl tRNA synthetase and the anticodon stem-loop oligonucleotide. Thus, the anticodon binding domain is functionally independent of the class-defining catalytic core and can be joined to another protein with little change in RNA binding characteristics.
Gale
A J
AJ
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Schimmel
P
P
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002942:Circular Dichroism; D017931:DNA Primers; D004586:Electrophoresis / Q000379:methods; D029968:Escherichia coli Proteins; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009690:Nucleic Acid Conformation; D011489:Protein Denaturation; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases