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Research Paper
E. coli Glutamyl-tRNA Synthetase is Inhibited by Anticodon Stem-Loop Domains and a Minihelix
Estella M. Gustilo, Daniel Dubois, Jacques Lapointe, Daniel Dubois and Paul Agris
volume 4 | issue 2
april/may/june 2007Pages: 85 - 92
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Portions of E. coli tRNAGlu having identity determinants for glutamyl-tRNA synthetase (ERS, EC 6.1.1.17) have been designed to be the first RNA inhibitors of a Class I synthetase. ERS recognizes the 2-thionyl group of 2-thio-5-methylaminomethyluridine (mnm5s2U34) in the first or wobble anticodon position of E. coli tRNAGlu. The interaction, as revealed by structural analysis, though specific, appears tenuous. Thus, it is surprising that RNAs designed from this tRNA's anticodon stem and loop domain with (ASLGlu-s2U34) and without s2U34 are bound by ERS and inhibit aminoacylation of the native tRNA. ASLGlu, ASLGlu-s2U34, and a minihelixGlu composed of identity determinants of the amino acid accepting stem were thermally stable under conditions of aminoacylation (Tms = 75 ± 1.5, 76 ± 0.9 and 83 ± 2.0 °C, respectively). In binding competition, the modified ASLGlu-s2U34 bound ERS with a higher affinity (half maximal inhibiting concentration, IC50, 5.1 ± 0.4 µM) than its unmodified counterpart, ASLGlu (IC50, 10.3 ± 0.6 µM). The minihelixGlu, ASLGlu-s2U34, and ASLGlu bound ERS with Kds of 9.9 ± 3.3, 6.5 ± 1.7 and 20.5 ± 3.8 µM. ERS aminoacylation of tRNAGlu was inhibited by the tRNA fragments. Unmodified ASLGlu, minihelixGlu, and ASLGlu-s2U34 exhibited a Kic of 1.9 ± 0.2 µM, 4.1 ± 0.2 µM, and 6.5 ± 0.1 µM, respectively. The modified ASLGlu-s2U34, though having a higher affinity for ERS, may be released more readily and thus, not be as good an inhibitor as the unmodified ASL. Thus, the RNA constructs are effective tools to study RNA-protein interaction.
Authors
Estella M. Gustilo
North Carolina State University; Raleigh, NC
Daniel Dubois
Université Laval; Québec, Canada
Jacques Lapointe
Universite Laval
Daniel Dubois
Université Laval; Québec, Canada
Paul Agris
North Carolina State University; Raleigh, NC
We now provide open access to journal articles published online for one year or more. This article may be downloaded at the following link:
Download PDF If the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.