Probing of metal-binding domains of RNA hairpin loops by laser-induced lanthanide(III) luminescence.
Direct laser excitation of aqueous Eu(III) bound to specific RNA fragments was used to probe the metal-binding sites of the anticodon loop of tRNA(Phe) from E. coli and of a tetraloop containing a GNRA consensus sequence. Binding of Mg(II) or Eu(III) to either RNA fragment resulted in a higher melting transition, but no global change in structure was observed. Aqueous Eu(III) exhibits a single weak excitation peak at 17273 cm(-1), the intensity of which increased upon addition of the tRNA loop fragment. Analysis of incremental increases in the luminescence intensity upon complexation with the tRNA loop indicated a stoichiometry of one high-affinity Eu(III)-binding site per loop fragment, with a Kd of 1.3 +/- 0.2 microM. Competition experiments between Eu(III) and Mg(II) were consistent with the two metal ions binding to a common site and with an approximately 30-fold lesser affinity of the tRNA loop for Mg(II) than for Eu(III). The rate of luminescence decay following excitation of Eu(III) bound to the tRNA loop corresponded to displacement of up to 4-5 (of a possible 9) waters of hydration on binding to the tRNA loop. By comparison, Eu(III) binds to the DNA analogue of the tRNA loop with an 8-fold lesser affinity and one fewer direct coordination site than to the RNA sequence, suggesting that a 2'OH of RNA is one of the direct ligands. In contrast with the absence of a shift in the excitation peak of aqueous Eu(III) upon formation of the tRNA loop complex, direct excitation of Eu(III) bound to a GNRA tetraloop fragment resulted in a substantially blue-shifted excitation peak (17290 cm(-1)). The tetraloop fragment also has a single Eu(III)-binding site, with a Kd of 12 +/- 3 microM. The bound Eu(III) was competed by Mg(II), although the relative affinity for Mg(II) was approximately 150-450-fold less than that for Eu(III). The Eu(III)-binding site of the tetraloop site is highly dehydrated, with approximately 7 water molecules displaced upon binding by RNA ligands, suggesting that the blue-shift of the excitation peak is the result of Eu(III) specifically bound in a nonpolar site within the GNRA loop structure.
Greenbaum
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NL
Department of Chemistry, Florida State University, Tallahassee, Florida 32306-4390, USA. nancyg@chem.fsu.edu
Mundoma
C
C
Peterman
D R
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D000926:Anticodon / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002942:Circular Dichroism; D005063:Europium / Q000378:metabolism; D007811:Lanthanum / Q000378:metabolism; D007834:Lasers; D008163:Luminescent Measurements; D008274:Magnesium / Q000378:metabolism; D008674:Metals, Rare Earth / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D012996:Solutions; D015019:Yttrium
D000926:Anticodon; D008674:Metals, Rare Earth; D012329:RNA, Bacterial; D012360:RNA, Transfer, Phe; D012996:Solutions; D005063:Europium; D015019:Yttrium; D007811:Lanthanum; D008274:Magnesium