Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops.

We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.     

Factors affecting thermodynamic stabilities of RNA 3 x 3 internal loops

Internal loops in RNA are important for folding and function. The 3 x 3 nucleotide internal loops are the smallest size symmetric loops with a potential noncanonical base pair (middle pair) flanked on both sides by a noncanonical base pair (loop-terminal pair). Thermodynamic and structural insights acquired for 3 x 3 loops should improve approximations for stabilities of 3 x 3 and larger internal loops. Most natural 3 x 3 internal loops are purine rich, which is also true of other internal loops. A series of oligoribonucleotides containing different 3 x 3 internal loops were studied by UV melting and imino proton NMR. Both loop-terminal and middle pairs contribute to the thermodynamic stabilities of 3 x 3 loops. Extra stabilization of -1.2 kcal/mol was found for a GA middle pair when flanked by at least one non-pyrimidine-pyrimidine loop-terminal pair. A penalty of approximately 1 kcal/mol was found for loops with a single loop-terminal GA pair that has a U 3' to the G of the GA pair. A revised model for predicting stabilities of 3 x 3 loops is derived by multiple linear regression.     

Thermodynamics-structure relationship of single mismatches in RNA/DNA duplexes

Thermodynamics of 66 RNA/DNA duplexes containing single mismatches were measured by UV melting methods. Stability enhancements for rG. dT mismatches were the largest of all mismatches examined here, while rU.dG mismatches were not as stable. The methyl group on C5 of thymine enhanced the stability by 0.12 approximately 0.53 kcal mol(-)(1) depending on the identity of adjacent Watson-Crick base pairs, whereas the 2'-hydroxyl group in ribouridine stabilized the duplex by approximately 0.6 kcal mol(-)(1) regardless of the adjacent base pairs. Stabilities induced by the methyl group in thymine, the 2'-hydroxyl group of ribouridine, and an nucleotide exchange at rG.dT and rU.dG mismatches were found to be independent of each other. The order for the mismatch stabilities is rG.dT > rU. dG approximately rG.dG > rA.dG approximately rG.dA approximately rA. dC > rA.dA approximately rU.dT approximately rU.dC > rC.dA approximately rC.dT, although the identity of the adjacent base pairs slightly altered the order. The pH dependence stability and structural changes were suggested for the rA.dG but not for rG.dA mismatches. Comparisons of trinucleotide stabilities for G.T and G.U pairs in RNA, DNA, and RNA/DNA duplexes indicate that stable RNA/DNA mismatches exhibit a stability similar to RNA mismatches while unstable RNA/DNA mismatches show a stability similar to that of DNA mismatches. These results would be useful for the design of antisense oligonucleotides.     

Genetic analysis of the histidine operon control region of Salmonella typhimurium

Mutants of the histidine operon control region (hisO) include two classes: (1) those completely unable to express the operon (His auxotrophs), and (2) prototrophs that are unable to achieve fully induced levels of operon expression (still His⁺ but sensitive to the drug amino-triazole). Using new, as well as previously existing hisO mutants, we constructed a fine-structure deletion map of hisO. Mutations that presumably alter the his promoter map at one end of hisO; mutations that alter the his attenuator map at the other end of hisO. Between the promoter and the attenuator lie a number of mutations that affect either the translation of the his leader peptide gene, or the formation and stability of his leader messenger RNA structures. All of the point mutations mapping in this central region revert to His⁺ at a very high frequency (10^?5 to 10^?6); this frequency is increased by both base substitution and frameshift-inducing mutagens. Many of the His^? mutants are suppressed by informational suppressors; all three types of nonsense mutations have been identified, demonstrating that translation of a region of hisO between the promoter and attenuator is essential for his operon expression. All of the hisO mutations tested are cis-dominant.     

Calorimetric analysis of thermodynamic stability and aggregation for apo and holo amyotrophic lateral sclerosis-associated Gly-93 mutants of superoxide dismutase

Differential scanning calorimetry was used to measure changes in thermodynamic stability and aggregation for glycine 93 mutants of human copper, zinc-superoxide dismutase (SOD). Glycine 93 is a conserved residue at position i + 3 of a tight turn and has been found to be a mutational hot spot in familial amyotrophic lateral sclerosis (fALS). The fALS-associated mutations, G93A, G93S, G93R, G93D, and G93V, were made in a pseudo wild-type background containing no free cysteines, which prevented the formation of aberrant disulfide bonds upon thermal unfolding, and enabled quantitative thermodynamic analysis of the effects of the mutations. Thermal unfolding was highly reversible for all the SODs in both the fully metallated (holo) and metal-free (apo) forms. The data for all the holo-SODs and for the apo-pseudo-wild-type SOD were well fit by a 2-state unfolding model for native dimer (N2) to two unfolded monomers (2U), N2 <--> 2U. The holo- and apo-forms of the mutants are significantly destabilized (by 1.5-3.5 kcal mol(-1) monomer) relative to the corresponding forms of pseudo wild-type, with the relative stabilities being correlated with statistical preferences for amino acids in this structural context. Although van't Hoff (DeltaHvH) to calorimetric (DeltaHcal) enthalpy ratios are close to unity for all the holo-SODs and for apo-pseudo-wild-type, consistent with a 2-state transition, DeltaHvH is considerably larger than DeltaHcal for all the apo-mutants. This suggests that the mutations cause apo-SOD to have an increased propensity to misfold or aggregate, which may be linked to increased toxic mutant SOD aggregation in fALS.     

Thermodynamic characterization of single mismatches found in naturally occurring RNA

Many naturally occurring RNA structures contain single mismatches. However, the algorithms currently used to predict RNA structure from sequence rely on a minimal set of data for single mismatches, most of which occur rather infrequently in nature. As a result, several approximations and assumptions are used to predict the stability of RNA duplexes containing the most common single mismatches. Therefore, the relative frequency of single mismatches was determined by compiling and searching a database of 955 RNA secondary structures. Thermodynamic parameters for duplex formation, derived from optical melting experiments, are reported for 28 oligoribonucleotides containing frequently occurring single mismatches. These data were then combined with previous data to construct a dataset of 64 single mismatches, including the 30 most common in the database. Because of this increase in experimental thermodynamic parameters for single mismatches that occur frequently in nature, more accurate free energy calculations have resulted. To improve the prediction of the thermodynamic parameters for duplexes containing single mismatches that have not been experimentally measured, single mismatch-specific nearest neighbor parameters were derived. The free energy of an RNA duplex containing a single mismatch that has not been thermodynamically characterized can be calculated by: DeltaG degrees 37,single mismatch = DeltaG degrees 37,mismatch nt + DeltaG degrees 37,mismatch-NN interaction + DeltaG degrees 37,AU/GU. Here, DeltaG degrees 37,mismatch is -0.4, -2.1, and -0.3 kcal/mol for A.G, G.G, and U.U mismatches, respectively; DeltaG degrees 37,mismatch-NN interaction is 0.7, -0.5, 0.4, -0.4, and -1.0 kcal/mol for 5'YRR3'/3'RRY5', 5'RYY3'/3'YYR5', 5'YYR3'/3'RYY5', 5'YRY3'/3'RYR5', and 5'RRY3'/3'YYR5' mismatch-nearest neighbor combinations, respectively, when A and G are categorized as purines (R) and C and U are categorized as pyrimidines (Y); and DeltaG degrees 37,AU/GU is a penalty of 1.2 kcal/mol for replacing a G-C base pair with either an A-U or G-U base pair. Similar predictive models were also derived for DeltaH degrees single mismatch and DeltaS degrees single mismatch. These new predictive models, in conjunction with the reported thermodynamics for frequently occurring single mismatches, should allow for more accurate calculations of the free energy of RNA duplexes containing single mismatches and, furthermore, allow for improved prediction of secondary structure from sequence.     

Thermodynamic genetics of the folding of the B1 immunoglobulin-binding domain from streptococcal protein G

A method has been developed to select proteins that are thermodynamically destabilized yet still folded and functional. The DNA encoding the B1 IgG-binding domain from Group G Streptococcus (Strp G) has been fused to gene III of bacteriophage M13. The resulting fusion protein is displayed on the surface of the phage thus enabling the phage to bind to IgG molecules. In addition, these phage exhibit a small plaque phenotype that is reversed by mutations that destabilize the Strp G domain. By selecting phage with large plaque morphology that retain their IgG-binding function, it is possible to identify mutants that are folded but destabilized compared with wild-type Strp G. Such mutants can be divided into three general categories: (1) those that disrupt packing of hydrophobic side chains in the protein interior; (2) those that destabilize secondary structure; and (3) those that alter specific hydrogen bonds involving amino acid side chains. A number of the mutants have been physically characterized by circular dichroism and nuclear magnetic resonance and have been shown to have structures similar to wild-type Strp G but stabilities that were decreased by 2–5 kcal/mol. © 1995 Wiley-Liss, Inc.     

Physical and biological features of polyoma virus mutants able to infect embryonal carcinoma cell lines

Three new polyoma mutants were selected for their ability to grow on the embryonal carcinoma cell line F9. These mutants share in common an insertion of two nucleotides, a thymine and an adenine, in the noncoding region located on the late side of the origin of replication. We have found that these insertions exist in all of the other polyoma virus mutants able to grow on F9 cells (Fujimura et al., Cell 23:809-814, 1981; Katinka et al., Nature (London) 290:720-722, 1981; K. Sekikawa and A. J. Levine, Proc. Natl. Acad. Sci. U.S.A. 78:1100-1104, 1981). The region containing these insertions could be folded into a stable secondary structure which included a guanine plus cytosine (G + C)-rich stem. The adenine and thymine were inserted in such a way that they maintained the palindrome in the G + C-rich stem and were complementary in the putative secondary structure that we present here. Another class of polyoma virus mutants selected on a multipotential carcinoma cell line (PCC4-Aza) were characterized by a more complex rearrangement (deletion and duplication) which occurred in the same region. This arrangement preserved the G + C-rich palindrome and also yielded a sequence which still allowed the folding of another type of stable secondary structure. The significance of these findings is discussed.     

RNA hairpin loop stability depends on closing base pair.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXAUAAUAYCC, where X and Y are CG, GC, AU, UA, GU, or UG. A nearest neighbor analysis of the data indicates the free energy change for loop formation at 37 degrees C, delta degrees Gl,37, averages 3.4 kcal/mol for hairpin loops closed with C.G, G.C, and G.U pairs. In contrast, delta G degree l,37 averages 4.6 kcal/mol for loops closed with A.U, U.A, or U.G pairs. Thus the stability of an RNA hairpin depends on the closing base pair. The hairpin with a GA mismatch that is formed by GGCGUAAUAGCC is more stable than the corresponding hairpin with an AA mismatch. Thus hairpin stability also depends on loop sequence. These effects are not included in current algorithms for prediction of RNA structure from sequence.     

Contribution of the intercalated adenosine at the helical junction to the stability of the gag-pro frameshifting pseudoknot from mouse mammary tumor virus

The mouse mammary tumor virus (MMTV) gag-pro frameshifting pseudoknot is an H-type RNA pseudoknot that contains an unpaired adenosine (A14) at the junction of the two helical stems required for efficient frameshifting activity. The thermodynamics of folding of the MMTV vpk pseudoknot have been compared with a structurally homologous mutant RNA containing a G x U to G-C substitution at the helical junction (U13C RNA), and an A14 deletion mutation in that context (U13CdeltaA14 RNA). Dual wavelength optical melting and differential scanning calorimetry reveal that the unpaired adenosine contributes 0.7 (+/-0.2) kcal mol(-1) at low salt and 1.4 (+/-0.2) kcal mol(-1) to the stability (deltaG(0)37) at 1 M NaCl. This stability increment derives from a favorable enthalpy contribution to the stability deltadeltaH = 6.6 (+/-2.1) kcal mol(-1) with deltadeltaG(0)37 comparable to that predicted for the stacking of a dangling 3' unpaired adenosine on a G-C or G x U base pair. Group 1A monovalent ions, NH4+, Mg2+, and Co(NH3)6(3+) ions stabilize the A14 and deltaA14 pseudoknots to largely identical extents, revealing that the observed differences in stability in these molecules do not derive from a differential or specific accumulation of ions in the A14 versus deltaA14 pseudoknots. Knowledge of this free energy contribution may facilitate the prediction of RNA pseudoknot formation from primary nucleotide sequence (Gultyaev et al., 1999, RNA 5:609-617).