Dependence of tRNA Structure in Solution upon Ionic Condition of the Solvent

Abstract

Barley phenylalanine tRNA (tRNAPhe) exhibits a 4.7-fold enhancement of fluorescence intensity in response to the addition of MgClz in 0.01 M Tris-HC1, pH 7.5,O.OOl M Naz EDTA. In the case of 2.0 M NaCl about 60% of this effect was observed. The change of fluorescence intensity was used as a measure of bound ligand concentration. From fluorescence Scatchard plots a different character of Mg2+ binding to tRNA was revealed, depending upon the initial conformation of tRNA in solution submitted to titration. In NaC1-free tRNAPhe solution strong interacting (K, = 1.5 x lo5 M-', n = 2.4) and weak independent (K, = 9 x lo3 M-') Mg2+ binding were observed. In solution containing 0.1 M NaCl both the strong and the weak Mg2+ binding demonstrated independent behaviour. K, and K,, in the presence of NaCl, had lower values than in NaC1-free solution, since Na+ was found to be a competitive inhibitor of M$+ binding. The significance of cations for tRNA conformation and biological activity is well established by studies of divalent cation binding to tRNA and of their effect on tRNA structure in solution ([l], for reviews see [2,3]). Recently the conformational effects of monovalent cations have been also well documented [3 -101. Less attention has been given to the simultaneous effect of mono and divalent cations on the tRNA molecule in solution, although studies of this effect give closer approximation of the applied experimental conditions to the physiological ones. From results obtained by Goldstein et al. [3] it could be assumed that the differences between the conformational effect of Na' and Mgz+ are mainly of a quantitative character. However, as recently shown [ l l ] the simultaneous effect of Na+ and M$+ on tRNA is not a simple sum of the effects of these ions observed separately. Studies of divalent cation binding to tRNA were performed by equilibrium dialysis [12,13] and other methods [13 -161 allowing for direct determination of bound and free ligand concentration at equilibrium conditions. Using these 'direct methods' the following parameters describing the binding process were obtained : the number of classes of binding sites and their independent or interacting character, the binding constant and number of binding sites within a class of binding sites. On the other hand, measurements of changes in some properties of macromolecule (e.g. fluorescence intensity) as a function of ligand concentration also reveal information concerning ligand binding to the macromolecule. Assuming that 100 % of the observed parameter change, at ligand concentration approaching infinity, reflects saturation of macromolecule with ligand, it is possible to determine the fraction of the macromolecules with occupied binding sites at any given ligand concentration. These methods of ligand binding measurements, which are called here 'indirect methods' have been already applied for studies of cation binding to tRNA [9]. Applying indirect methods, we measure the effect of ligand binding and thus, the observations are limited to those binding...

Magnesium ion inner sphere complex in the anticodon loop of tRNAPhe

“…In the absence of Mg2+, these conformations are almost equally populated. According to various arguments (Labuda & Porschke, 1980;Urbanke & Maass, 1978), the transition probably corresponds to the one described by Fuller & Hodgson (1967). From the X-ray analysis, it is apparent that the Mg2+ ion by its many contacts to various bases strongly favors the 3'-stacked conformation.…”

“…By this program, the relaxation signals were corrected for the heating time, the rise time of the photomultiplier (a 5-ps rise time was used in all measurements), and the cutoff of the first part of the signal (about half of the amplitude corresponding to the unspecific fluorescence quench associated with the temperature change; cf. Labuda & Porschke, 1980).…”

“…Temperature-jump experiments were carried out essentially as described previously (Labuda & Porschke, 1980), using the same equipment (Rigler et al, 1974). This time, however, we used a Hanovia 600 W Hg/Xe lamp as a light source and a single Schoeffel GM-250 monochromator for excitation of the sample at 313 nm.…”

“…If the contacts are favorable, it should be possible to provide evidence for them by experiments in free solution. Actually recent relaxation experiments (Labuda & Porschke, 1980) showed an association of tRNAphe, which was induced by binding of the codon UUC. In the present contribution, this reaction is analyzed quantitatively by measurements of the sedimentation equilibrium in an analytical ultracentrifuge.…”

Fluorimetric Study of the Complex between Yeast Phenylalanyl‐tRNA Synthetase and tRNAPhe

“…The early studies [l-31 showed that the YWye fluorescence is a very sensitive probe for the local conformation of the anticodon loop and that it strongly depends upon external conditions, especially the Mg2+ concentration and pH. Studies of the YWye fluorescence intensity in barley tRNAPhe [4,5] and in yeast tRNAPhe [6], as a function of divalent cation concentration (respectively Mg2 + and Mn2 +) and monovalent cations, afforded an insight into the role of cations in the structure of tRNAs.Abbreviations. The modified base at position 37 of tRNAPhe from S. cerevisiae (formerly called Y base) is called wybutine, abbreviated YWye, as proposed by W. E. Cohn; CD, circular dichroism.…”