Investigation of the thermal unfolding of secondary and tertiary structure in E. coli tRNA^fMet by high‐resolution nmr

Abstract

SynopsisThe high-resolution (300 MHz) proton nmr spectrum of E. coli tRNAfMet has been examined in 0.17M NaCl, with and without hIg2+, and at various temperatures. In light of recent studies of other E. coli tRNA and fragments of tRNAfMet, some low field (1 1-15 ppm) resonances previously assigned to secondary structure base pairs are reassigned to a tertiary structure Alr-S4U8 base pair and a protected uridine residue in the anticodon loop. These two resonances and other low field resonances which are assigned t o secondary structure base pairs are used to monitor the thermal unfolding of the molecule. In the absence of Mgz+ the tertiary structure base pair is present only to -45"C, but in the presence of Mg2+ it remains until at least 70°C. Analysis of the temperature dependence of other low field resonances indicates that the melting of the dihydrouridine stem occurs more or less simultaneously with the loss of tertiary structure. The observation of the resonance from the A14-S4U8 base pair proves that tertiary structure is present in this molecule below 40°C, even in the absence of Mgz+.

A spin label study of the thermal unfolding of secondary and tertiary structure in E.coli transfer RNAs

“…The molecular mechanism, proposed by Crothers et al (3), for the thermal unfolding of tRNAfMet, in the absence of magnesium, in such that at 300 part of the hU helix can open transiently, followed by the simultaneous disruption of "tertiary interactions" and the rest of the hU helix (460), and then, in succession, T*C (610), anticodon helix (700) and finally the acceptor stem (770). On the other hand, the NMR work of Kearns and coworkers (2) argues that, without magnesium, the hU stem and tertiary structure melt more or less simultaneously at -450 whereas the anticodon, TpC and amino acid acceptor stems melt between 560 and 70°In the presence of magnesium, however, they observed a perturbation of the spectra at 250, followed at 600 by the unfolding of hU and anti-codon regions and finally full denaturation occurs between 680 and 850.…”

“…Our results are compared with those obtained by other techniques giving insight into the secondary and tertiary structure of tRNA molecules in solution: NMR (2,3), fluorescence (4), temperature-jump relaxation kinetics (3) as well as thermodynamic (5) and calorimetric measurements (6) EXPERIMENTAL Materials and Methods All the spin-labeled tRNAs used in this study have been described in the preceeding paper (1). The EPR spectra were obtained under conditions such that the native structure of the tRNA species is assumed to be preserved (0.02 M Tris-HCl, pH 7.5 with 10 mM MgCl2).…”

Contribution of quantum-mechanical computations to the elucidation of the solution structure of tRNA by NMR techniques

“…It has been used for this purpose and for a few different tRNAs in different laboratories [2-151. Besides its direct significance for the problem treated here, the contribution of the theory has indirect repercussions on a group of problems depending upon the assignment of resonances to specific base pairs of tRNAs. Thus due to this assignment one may follow in detail the mcx?ications of the molecular structure of a tRNA under the influence of heat, the progressive disappearance of resonances with an increase of temperature being then linked to the rupture of specific base pairs (for example [9,241). Similarly, the effect of cations, for instance Mg++, on the spectrum of tRNAs may be interpreted in terms of…”

Intensity‐fluctuation spectroscopy and tRNA conformation. II. Changes of size and shape of tRNA in the melting process

“…The theory of correlation function measurements by time-of-arrival of photoelectron pulses has been de~eloped.~5,~6 Our system is similar in operational principle to that of Chopra and Mande1,28 except that we can measure (sample) delay time down to 1 nsec. At low count rate, No G(2)(7) = C Pc(l,l; 7 ; AT1i counts in (0,~)) (15) where Pc(l,l; 7; A71 i counts in (0,~)) is the conditional probability of having i counts within time interval (0,~) and No is the number of stops. Thus, for low count rates over very short delay times,…”