Thermodynamic Characterization of RNA Triloops

Abstract

Relatively few thermodynamic parameters are available for RNA triloops. Therefore, 24 stemloop sequences containing naturally occurring triloops were optically melted, and the thermodynamic parameters ΔH°, ΔS°, ΔG°3 7 , and T M for each stem-loop were determined. These new experimental values, on average, are 0.5 kcal/mol different from the values predicted for these triloops using the model proposed by Mathews et al. [Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl. Acad. Sci. USA 101, 7287-7292]. The data for the 24 triloops reported here were then combined with the data for five triloops that were published previously. A new model was derived to predict the free energy contribution of previously unmeasured triloops. The average absolute difference between the measured values and the values predicted using this proposed model is 0.3 kcal/mol. These new experimental data and updated predictive model allow for more accurate calculations of the free energy of RNA stem-loops containing triloops, and, furthermore, should allow for improved prediction of secondary structure from sequence.RNA stem-loops containing three nucleotides in the loop, triloops, are common secondary structure motifs found in naturally occurring RNA. For example, bacterial 16S rRNAs strongly favor tetraloops; however, the UUU triloop is the most common replacement (1). In the 16S-like rRNA variable regions, triloops account for 7% of the loops in bacteria and 16% of the loops in eukaryotes (2). Triloops are also found in large subunit rRNAs (3,4), 5S rRNAs (5), signal recognition particles (6), RNase P RNAs (7), and group I introns (8,9). More specifically, triloops are found in Brome mosaic virus (+) strand RNA (10), human rhinovirus isotype 14 (11), iron responsive element RNA (12), and an RNA aptamer for bacteriophage MS2 coat protein (13), to name a few. Although relatively unstable due to the strain in the loop, triloops may be an important structural feature due to the accessibility of the loop nucleotides for recognition by proteins, other nucleic acids, or small molecules. It has been shown that triloops play a role in various biological processes, including virus replication (11,14), viral synthesis (15), and iron response (12), to name a few. Nevertheless, only a few studies have thermodynamically characterized RNA triloops (16)(17)(18)(19)(20), and only three of these (16-18) were done using 1 M NaCl (the salt concentration in which most nearest neighbor parameters are derived).The current model used by secondary structure prediction algorithms to predict the thermodynamic contribution of RNA triloops to stem-loop stability is sequence independent; all triloops contribute 5.4 kcal/mol to stem-loop stability, with the exception of 5'CCC3' which contributes 6.9 kcal/mol (21). In addition, there are two unstable triloops sequences (5'CAACG3' and 5'GUUAC3') for which this predictive model is not used; instead, the ΔG°3 7,loop values (6.8 and 6.9 kcal/mol, respecti...

Structural and thermodynamic signatures that define pseudotriloop RNA hairpins

“…The intensity of this resonance in a number of different loop sequences correlates well with their measured thermodynamic stabilities. We conclude that PTL stability depends on (i) stacking energies between cross-loop and loop-closing base pairs, with CG and GC bp, respectively, resulting in the highest stability; (ii) identity of the 3 ′ bulge, with pyrimidines favoring and purines disfavoring PTL formation; (iii) sequence in the resulting triloop, where the same trend in stability is observed as for regular triloops (Shu and Bevilacqua 1999;Thulasi et al 2010). The latter observation suggests that incorporation of a bonus for certain stable triloop motifs in RNA folding algorithms will likely result in the prediction of more PTLs as well.…”

“…Also the other sequences with loop-closing CG base pair, tHIV and tPYR, showed comparable low ΔG°3 7 s ranging from −6.2 to −6.6 kcal/mol, but lacked a crossloop base pair. Apparently the closing CG base pair in these cases stabilizes hexaloop formation as shown before (Serra et al 1993) and in triloops (Shu and Bevilacqua 1999;Thulasi et al 2010) and tetraloops (Antao et al 1991;Antao and Tinoco 1992;Blose et al 2009) but counteracts formation of a PTL.…”

The role of short RNA loops in recognition of a single-hairpin exon derived from a mammalian-wide interspersed repeat

“…Finally, the higher exon inclusion of G(NNN)C triloops as compared to their C(NNN)G counterparts (Fig. S5) may reflect a previously described lower thermodynamic stability of the former 48 and contribute to the observed positive correlation between splicing activity of the hairpin and free energy (Fig. 1E).…”

“…To begin to answer this question, we compared frequencies of 823 triloops in previously determined 1349 RNA secondary structures and the MIR exon inclusion of matching triloop mutants. The triloop database was derived from structures of 123 small subunit rRNAs, 223 large subunit rRNAs, 309 5S rRNAs, 484 tRNAs, 91 signal recognition particles, 16 RNase P RNAs, 100 group I introns and 3 group II introns 48 (Brent Znosko, personal communication). Interestingly, triloops frequent in natural RNAs tended to have low exon inclusion values and vice versa (Fig.…”

“…Triloop frequencies of previously determined RNA stem-loop structures were determined for a total of 823 triloops in 123 small subunit rRNAs, 223 large subunit rRNAs, 309 5S rRNAs, 484 tRNAs, 91 signal recognition particles, 16 RNase P RNAs, 100 group I introns and 3 group II introns 48 (and Brent Znosko, personal communication). The triloop database was considered to be representative of triloops found in naturally occurring RNAs.…”