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A novel 5′ displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA

Published online by Cambridge University Press:  02 March 2001


J.C. MACOSKO
Affiliation:
Department of Chemistry, University of California, Berkeley, and Structural Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720-1460, USA
M.S. PIO
Affiliation:
Department of Chemistry, University of California, Berkeley, and Structural Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720-1460, USA
I. TINOCO
Affiliation:
Department of Chemistry, University of California, Berkeley, and Structural Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720-1460, USA
Y.-K. SHIN
Affiliation:
Department of Chemistry, University of California, Berkeley, and Structural Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720-1460, USA

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Abstract

An RNA spin-labeling technique was developed using the well-characterized interaction between the HIV Rev peptide and the Rev response element (RRE) RNA as a model system. Spin-labeled RNA molecules were prepared by incorporating guanosine monophosphorothioate (GMPS) at the 5′ end using T7 RNA polymerase and then covalently attaching a thiol-specific nitroxide spin label. Three different constructs of the RRE RNA were made by strategically displacing the 5′ end within the native three-dimensional structure. Nitroxide-to-nitroxide distance measurements were made between the specifically bound RNA and peptide using electron paramagnetic resonance (EPR) spectroscopy. The dipolar EPR method can reliably measure distances up to 25 Å, the calculation of which is derived from the 1/r3 dependence of the broadening of EPR lines in motionally frozen samples. This RNA-labeling technique, dubbed 5′ displacement spin labeling, extends the usefulness of the dipolar EPR method developed for analysis of protein structure. The advantage of this technique is that it is applicable to large RNA systems such as the ribosome, which are difficult to study by other structural methods.


Type
Research Article
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Copyright
© 1999 RNA Society

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