Tertiary core rearrangements in a tight binding transfer RNA aptamer.
Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5'-44CAUUC48-3' is replaced by 5'-44AGGU48-3'. This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type. The 2.7 A cocrystal structure of the RNA aptamer-GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA-protein interface identical to the wild type. The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15-U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases. These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA-protein contacts.
Bullock
T L
TL
Department of Chemistry and Biochemistry and Interdepartmental Program in Biochemistry and Molecular Biology, University of California at Santa Barbara, 93106-9510, USA.
Sherlin
L D
LD
Perona
J J
JJ
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000378:metabolism*; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012351:RNA, Transfer, Gln / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D013816:Thermodynamics
D009843:Oligoribonucleotides; D012351:RNA, Transfer, Gln; D016601:RNA-Binding Proteins; D013431:Sulfates; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase