Dynamics of the RNA hairpin GNRA tetraloop

The dynamics of RNA hairpin tetraloops of the GNRA type [sequence G- any ri bonucleotide (N)-purine (R)-A] was analyzed by fluorescence spectroscopy an d by fluorescence-detected temperature-jump relaxation, using RNA oligomers with 2-aminopurine (2AP) substituted in two different positions of the loo p sequence, Gp2APpApA (HP1) and GpAp2APpA (HP2), as indicator. The fluoresc ence of HP1 is much higher than that of HP2, indicating a lower degree of 2 AP-stacking in HP1. Addition of Mg2+ or Ca2+ ions leads to an increase of f luorescence in HP1, whereas a decrease of fluorescence is observed in HP2, In both cases at least two ion-binding equilibria are required to fit titra tion data, T-jump experiments using fluorescence detection show a relaxatio n process with a time constant of 22 mu s for HP1, whereas two relaxation p rocesses with time constants 5 and 41 mu s, are found for HP2, These result s clearly demonstrate the existence of more than the single conformation st ate detected by NMR analysis. The T-jump amplitudes decrease with increasin g bivalent ion concentration, indicating that one of the states is favored in the presence of bivalent ions. The loop relaxation processes are slower than standard stacking processes, probably because of activation barriers i mposed by a restricted mobility of loop residues, and are assigned to a sta cking rearrangement, probably between the 5' and the 3'-side. A similar pro cess has been observed previously for the anticodon loop of tRNA(Phe). The rate constants of the transition are in the range of 10(4) s(-1) in the cas e of HP1. The data demonstrate the existence of structures that are not res olved by standard NMR because of fast exchange and are not found by X-ray a nalysis because of restrictions by crystal packing.