Anticodon loop of tRNAPhe: structure, dynamics, and Mg2+ binding.
The structure, dynamics, and Mg2+ binding reactions of the isolated anticodon hairpin loop from tRNAPhe (yeast) have been analyzed by fluorescence-detected temperature-jump relaxation, melting experiments, and equilibrium sedimentation. Most of the measurements were performed at an ionic strength of 0.15 M and at temperatures below 25 degrees C, where the hairpin loop proved to be stable. A relaxation effect with a time constant of approximately 100 microseconds, indicated by the Wye base fluorescence, is attributed to a conformational change of the anticodon loop and is very similar to a corresponding transition observed previously for the whole tRNAPhe molecule. A Mg2+ binding site reflected by an inner-sphere relaxation process and associated with a strong increase of the Wye base fluorescence closely resembles a corresponding site observed in the complete tRNAPhe and is attributed to a site in the anticodon loop identified by X-ray analysis. In addition to the Mg2+ site in the loop, which is associated with a binding constant of 2 X 10(3) M-1, the existence of sites with a higher affinity is demonstrated by an unusual relaxation effect, showing a minimum in the reciprocal time constant with increasing Mg2+ concentration. The experimental data can be described by a transition between two states and Mg2+ binding to both states resulting in a reaction cycle, which is extended by an additional Mg2+ binding reaction to one of the states. The unusual effect has not been observed for the complete tRNAPhe and is also not observed when Ca2+ is added instead of Mg2+. This result indicates the existence of a conformational change involving Mg2+ inner-sphere complexation.(ABSTRACT TRUNCATED AT 250 WORDS)
D000926:Anticodon; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013050:Spectrometry, Fluorescence
D000926:Anticodon; D012346:RNA, Transfer, Amino Acyl; D012343:RNA, Transfer; D008274:Magnesium