title: Biostructural chemistry of magnesium ion: characterization of the weak binding sites on tRNA(Phe)(yeast). Implications for conformational change and activity.
abstract: The thermodynamics and kinetics of magnesium binding to tRNA(Phe)(yeast) have been studied directly by 25Mg NMR. In 0.17 M Na+(aq), tRNA(Phe) exists in its native conformation and the number of strong binding sites (Ka greater than or equal to 10(4)) was estimated to be 3-4 by titration experiments, in agreement with X-ray structural data for crystalline tRNA(Phe) (Jack et al., 1977). The set of weakly bound ions were in slow exchange and 25Mg NMR resonances were in the near-extreme-narrowing limit. The line shapes of the exchange-broadened magnesium resonance were indistinguishable from Lorentzian form. The number of weak magnesium binding sites was determined to be 50 +/- 8 in the native conformation and a total line-shape analysis of the exchange-broadened 25 Mg2+ NMR resonance gave an association constant Ka of (2.2 +/- 0.2) X 10(2) M-1, a quadrupolar coupling constant (chi B) of 0.84 MHz, an activation free energy (delta G*) of 12.8 +/- 0.2 kcal mol-1, and an off-rate (koff) of (2.5 +/- 0.4) X 10(3) s-1. In the absence of background Na+(aq), up to 12 +/- 2 magnesium ions bind cooperatively, and 73 +/- 10 additional weak binding sites were determined. The binding parameters in the nonnative conformation were Ka = (2.5 +/- 0.2) X 10(2) M-1, chi B = 0.64 MHz, delta G* = 13.1 +/- 0.2 kcal mol-1, and koff = (1.6 +/- 0.4) X 10(3) s-1. In comparison to Mg2+ binding to proteins (chi B typically ca. 1.1-1.6 MHz) the lower chi B values suggest a higher degree of symmetry for the ligand environment of Mg2+ bound to tRNA. A small number of specific weakly bound Mg2+ appear to be important for the change from a nonnative to a native conformation. Implications for interactions with the ribosome are discussed.
authors.affiliation: Evans Laboratory of Chemistry, Ohio State University, Columbus 43210.
authors.forename: S S
authors.initials: SS
authors.lastname: Reid
authors.forename: J A
authors.initials: JA
authors.lastname: Cowan
mesh_terms: D001665:Binding Sites; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009690:Nucleic Acid Conformation; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
chemical_list: D012345:RNA, Transfer, Amino Acid-Specific; D012360:RNA, Transfer, Phe; D008274:Magnesium