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Specific metal-ion binding sites in a model of the P4-P6 triple-helical domain of a group I intron

Published online by Cambridge University Press:  27 July 2001

MARTINA LINDQVIST ,
KARIN SANDSTRÖM ,
VILNIS LIEPINS ,
ROGER STRÖMBERG  and
ASTRID GRÄSLUND
MARTINA LINDQVIST
Affiliation:
Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden
KARIN SANDSTRÖM
Affiliation:
Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden
VILNIS LIEPINS
Affiliation:
Division of Organic and Bioorganic Chemistry, Department of Medical Biochemistry and Biophysics, Scheele Laboratory, Karolinska Institutet, S-17177 Stockholm, Sweden
ROGER STRÖMBERG
Affiliation:
Division of Organic and Bioorganic Chemistry, Department of Medical Biochemistry and Biophysics, Scheele Laboratory, Karolinska Institutet, S-17177 Stockholm, Sweden
ASTRID GRÄSLUND
Affiliation:
Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden
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Abstract

Divalent metal ions play a crucial role in RNA structure and catalysis. Phosphorothioate substitution and manganese rescue experiments can reveal phosphate oxygens interacting specifically with magnesium ions essential for structure and/or activity. In this study, phosphorothioate interference experiments in combination with structural sensitive circular dichroism spectroscopy have been used to probe molecular interactions underlying an important RNA structural motif. We have studied a synthetic model of the P4-P6 triple-helical domain in the bacteriophage T4 nrdB group I intron, which has a core sequence analogous to the Tetrahymena ribozyme. Rp and Sp sulfur substitutions were introduced into two adjacent nucleotides positioned at the 3′ end of helix P6 (U452) and in the joining region J6/7 (U453). The effects of sulfur substitution on triple helix formation in the presence of different ratios of magnesium and manganese were studied by the use of difference circular dichroism spectroscopy. The results show that the pro-Sp oxygen of U452 acts as a ligand for a structurally important magnesium ion, whereas no such effect is seen for the pro-Rp oxygen of U452. The importance of the pro-Rp and pro-Sp oxygens of U453 is less clear, because addition of manganese could not significantly restore the triple-helical interactions within the isolated substituted model systems. The interpretation is that U453 is so sensitive to structural disturbance that any change at this position hinders the proper formation of the triple helix.

Keywords

difference CD spectroscopyphosphorothioate interferenceRNA-metal binding
Type
Research Article
Information
RNA , Volume 7 , Issue 8 , August 2001 , pp. 1115 - 1125
Copyright
© 2001 RNA Society

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