We have used high resolution NMR and thermodynamics to characterize the secondary structure and stability of the selenocysteine insertion sequences (SECIS) of human glutathione peroxidase (58 nt) and thioredoxin reductase (51 nt). These sequences are members of the two classes of SECIS recently identified with two distinct structures capable of directing selenocysteine incorporation into proteins in eukaryotes. UV melting experiments showed a single cooperative and reversible transition for each RNA, which indicates the presence of stable secondary structures. Despite their large size, the RNAs gave well resolved NMR spectra for the exchangeable protons. Using NOESY, the imino protons as well as the cytosine amino protons of all of the Watson-Crick base pairs were assigned. In addition, a number of noncanonical base pairs including the wobble G.U pairs were identified. The interbase-pair NOEs allowed definition of the hydrogen-bonded structure of the oligonucleotides, providing an experimental model of the secondary structure of these elements. The derived secondary structures are consistent with several features of the predicted models, but with some important differences, especially regarding the conserved sequence motifs.
|Number of pages||10|
|Journal||Nucleic Acids Research|
|State||Published - 2004|
Bibliographical noteFunding Information:
We thank Dr G. Kelley and Dr T.A. Frenkiel for advice and Dr A. Pastore for encouragement. NMR spectra were recorded at the MRC NMR Centre, Mill Hill, and at the James Graham Brown NMR Center, University of Louisville. This work was supported in part by the MRC (UK), the Brown Foundation (USA) and NCI grant 1R01CA101199. A.N.L. gratefully acknowledges the Kentucky Challenge for Excellence Program.
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