RNA. 1999 Sep;5(9):1158-66. Unique Identifier : AIDSLINE MED/99424536
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 A, 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.
JOURNAL ARTICLE Electron Spin Resonance Spectroscopy/*METHODS
Electrophoresis, Polyacrylamide Gel Genes, env/GENETICS Models,
Genetic Models, Theoretical Mutagenesis Nuclear Magnetic Resonance
Peptide Synthesis RNA/*ANALYSIS/CHEMICAL SYNTHESIS *Spin Labels
Support, U.S. Gov't, Non-P.H.S. Support, U.S. Gov't, P.H.S.