Optical melting was used to determine the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concentration. The oligomers included helices composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concentration on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temperature or free energy) was three times greater than that observed for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes observed upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes observed in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the observed cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calculated.

The association and dissociation rate constants of eight different E. coli aminoacyl-tRNAs (aa-tRNAs) for E. coli ribosomes programmed with mRNAs of defined sequences were determined. Identical association and dissociation rate constants were observed for all eight aa-tRNAs in both the ribosomal A and P sites despite substantial differences in tRNA sequence, the type of esterified amino acid, and posttranscriptional modifications. These results indicate that the overall binding of all aa-tRNAs to the ribosome is uniform. However, when either the esterified amino acid or the tRNA modifications were removed, binding was no longer uniform. These results suggest that differences in tRNA sequences and tRNA modifications have evolved to offset differential thermodynamic contributions of the esterified amino acid and the codon-anticodon interaction so that ribosomal binding of all aa-tRNAs remains uniform.

When different mutations were introduced into the anticodon loop and at position 73 of YFA2, a derivative of yeast tRNA(Phe), a single tRNA body was misacylated with 13 different amino acids. The affinities of these misacylated tRNAs for Thermus thermophilus elongation factor Tu (EF-Tu).GTP were determined using a ribonuclease protection assay. A range of 2.5 kcal/mol in the binding energies was observed, clearly demonstrating that EF-Tu specifically recognizes the side chain of the esterified amino acid. Furthermore, this specificity can be altered by introducing a mutation in the amino acid binding pocket on the surface of EF-Tu. Also, when discussed in conjunction with the previously determined specificity of EF-Tu for the tRNA body, these experiments further demonstrate that EF-Tu uses thermodynamic compensation to bind cognate aminoacyl-tRNAs similarly.

The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes was substantially impaired, as compared with the binding of their natural tRNAs, when they were transplanted into tRNA(2)(Ala). An analysis of chimeras composed of tRNA(2)(Ala) and various amounts of either tRNA(3)(Gly) or tRNA(2)(Arg) indicates that the presence of the parental 32-38 nucleotide pair is sufficient to restore ribosome binding of the transplanted anticodons. Furthermore, mutagenesis of tRNA(2)(Ala) showed that its highly conserved A32-U38 pair serves to weaken ribosome affinity. We propose that this negative binding determinant is used to offset the very tight codon-anticodon interaction of tRNA(2)(Ala). This suggests that each tRNA sequence has coevolved with its anticodon to tune ribosome affinity to a value that is the same for all tRNAs.

No abstract

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXANmAYCC, where XY is the wobble base pair, GU or UG, and the underlined loop sequences are three to eight nucleotides. A nearest-neighbor analysis indicates the free energy of loop formation is dependent upon loop size and closing base pair. Hairpin loops closed by UG base pairs are on average 1.3 kcal/mol less stable than hairpins closed by GU base pairs. The hairpin loops closed by UG have approximately the same stability as hairpin loops closed by AU/UA base pairs, while the loops closed by GU are approximately 0.7 kcal/mol more stable than hairpins loops closed by GC/CG base pairs. These results, combined with the model previously developed [Serra et al. (1997) Biochemistry 36, 4844] to predict the stability for hairpin loops closed by Watson-Crick base pairs, allow for the following model to predict the stability of hairpin loops: delta G degree 37L(n) = delta G degree 37iL(n) + delta G degree 37mm + 0.6 (if closed by AU, UA, or UB) - 0.7 (if closed by GU) - 0.7 (if first mismatch is GA or UU except for loops closed by GU). Here, delta G degree 37iL(n) is the free energy increment for initiating a loop of n nucleotides with a CG or GC pair, and delta G degree 37mm is the free energy for the interaction of the first mismatch with the closing base pair. For hairpin loops of n = 4-9, delta G037iL(n) is 4.9, 5.0, 5.0, 5.0, 4.9, and 5.5 kcal/mol, respectively. For hairpin loops of n = 3, delta G degree 37L(3) = +4.8 + 0.6 (if closed by AU, UA, or UG) kcal/mol. Thermodynamic parameters for hairpin formation in 1 M NaCl for 13 naturally occurring RNA hairpin sequences closed by wobble base pairs are reported. The model provides good agreement for both TM and delta G degree 37 for most hairpins studied. Thermodynamic values for five terminal mismatches adjacent to wobble base pairs are also reported.

No abstract

No abstract