Magnesium ion inner sphere complex in the anticodon loop of phenylalanine transfer ribonucleic acid.
The binding of Ca2+ and Mg2+ to tRNAPhe is analyzed by equilibrium titrations and temperature-jump measurements using the Wye base fluorescence as a label. Titration experiments starting with the folded structure of the tRNA (high salt and low temperature) show that Ca2+ and Mg2+ binding detected by Wye base fluorescence changes is associated with equilibrium constants between 1 x 10(3) and 3 x 10(3) M-1. The binding of Ca2+ leads to an increase of the relaxation time associated with a conformation change of the anticodon loop and to a decrease of the corresponding amplitude. These data are represented quantitatively by a two-step reaction scheme with a preferential binding of Ca2+ to one of the anticodon conformations. When Mg2+ is added, an extra relaxation process is observed with time constants around 1 ms. This process demonstrates the formation of a Mg2+ inner sphere complex. Relaxation time constants and amplitudes are represented quantitatively by a three-step reaction scheme. Mg2+ binds preferentially to one of the anticodon conformations. In the absence of Mg2+, these conformations are populated almost equally with a transition rate constant around 5 x 10(3) s-1. The Mg2+ inner sphere complex is formed with a relatively low rate constant of (1--2) x 10(3) s-1, indicating a conformational barrier. These data strongly suggest that the Mg2+ site analyzed in the present investigation corresponds to the anticodon site with a distorted octahedral coordination characterized by X-ray analysis. The results are discussed in terms of the anticodon function and also with respect to their implications upon Mg2+ binding to nucleic acids in general.
D000926:Anticodon / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D005453:Fluorescence; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012331:RNA, Fungal / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D013816:Thermodynamics
D000926:Anticodon; D002413:Cations, Divalent; D012331:RNA, Fungal; D012346:RNA, Transfer, Amino Acyl; D008274:Magnesium; D002118:Calcium