Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme using luminescence spectroscopy.
BACKGROUND
Divalent metal ions serve as structural as well as catalytic cofactors in the hammerhead ribozyme reaction. The natural cofactor in these reactions is Mg(II), but its spectroscopic silence makes it difficult to study. We previously showed that a single Tb(III) ion inhibits the hammerhead ribozyme by site-specific competition for a Mg(II) ion and therefore can be used as a spectroscopic probe for the Mg(II) it replaces.


RESULTS
Lanthanide luminescence spectroscopy was used to study the coordination environment around Tb(III) and Eu(III) ions bound to the structurally well-characterized site on the hammerhead ribozyme. Sensitized emission and direct excitation experiments show that a single lanthanide ion binds to the ribozyme under these conditions and that three waters of hydration are displaced from the Tb(III) upon binding the RNA. Furthermore, we show that these techniques allow the comparison of binding affinities for a series of ions to this site. The binding affinities for ions at the G5 site correlates linearly with the function Z(2)/r of the aqua ion (where Z is the charge and r is the radius of the ion).


CONCLUSIONS
This study compares the crystallographic nature of the G5 metal-binding site with solution measurements and gives a clearer picture of the coordination environment of this ion. These results provide one of the best characterized metal-binding sites from a ribozyme, so we use this information to compare the RNA site with that of typical metalloproteins.
Feig
A L
AL
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
Panek
M
M
Horrocks
W D
WD
Uhlenbeck
O C
OC
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D005063:Europium / Q000737:chemistry* / Q000493:pharmacokinetics; D007700:Kinetics; D008163:Luminescent Measurements; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013237:Stereoisomerism; D013725:Terbium / Q000737:chemistry* / Q000493:pharmacokinetics
D016337:RNA, Catalytic; D013725:Terbium; D005063:Europium