Contribution of the Closing Base Pair to Exceptional Stability in RNA Tetraloops: Roles for Molecular Mimicry and Electrostatic Factors

Blose, Joshua M.; Proctor, David J.; Veeraraghavan, Narayanan; Misra, Vinod K.; Bevilacqua, Philip C.

doi:10.1021/ja900065e

Cited by 39 publications

(69 citation statements)

References 63 publications

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“…For both gCUUGc and cGCAAg, when the closing stem G is on the opposite side of the loop as the consensus loop G, the resulting RNA is measurably more stable than RNA sequences in which the stem G is on the same side (20). We observe that, for both cGCAAg and gCUUGc, a cross-stacked G-G interaction is able to preorganize the formation of the correct in-register upper stem base pair, which always forms before the lower stem base pair (Fig.…”

Section: Discussionmentioning

confidence: 99%

High-resolution reversible folding of hyperstable RNA tetraloops using molecular dynamics simulations

Chen

Garcı́a

2013

Proc. Natl. Acad. Sci. U.S.A.

284

497

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We report the de novo folding of three hyperstable RNA tetraloops to 1-3 Å rmsd from their experimentally determined structures using molecular dynamics simulations initialized in the unfolded state. RNA tetraloops with loop sequences UUCG, GCAA, or CUUG are hyperstable because of the formation of noncanonical loop-stabilizing interactions, and they are all faithfully reproduced to angstrom-level accuracy in replica exchange molecular dynamics simulations, including explicit solvent and ion molecules. This accuracy is accomplished using unique RNA parameters, in which biases that favor rigid, highly stacked conformations are corrected to accurately capture the inherent flexibility of ssRNA loops, accurate base stacking energetics, and purine syn-anti interconversions. In a departure from traditional quantum chemistrycentric approaches to force field optimization, our parameters are calibrated directly from thermodynamic and kinetic measurements of intra-and internucleotide structural transitions. The ability to recapitulate the signature noncanonical interactions of the three most abundant hyperstable stem loop motifs represents a significant milestone to the accurate prediction of RNA tertiary structure using unbiased all-atom molecular dynamics simulations.RNA folding | molecular simulations S tructured RNAs exhibit a distinct preference for loops of precisely 4 nt, which was originally noted by Woese et al. (1) using comparative sequence analysis of ribosomes. Approximately 70% of these tetraloops are comprised of just three specific loop sequences: UUCG, GCAA, or CUUG. The abundance of these sequences is thermodynamic in origin, because each motif forms a unique network of noncanonical interactions within their loops that stabilizes the folded state. The abundance of high-resolution structural and thermodynamic data available for these motifs coupled with their characteristic noncanonical signatures make them ideal for adjudicating the accuracy of RNA folding simulations.RNA folding is understood to be hierarchical in nature, with secondary and tertiary folds stabilized by distinct thermodynamic driving forces (2). Secondary structure (the formation of canonical helices stabilized by Watson-Crick base pairs) can be accurately predicted from the nucleotide sequence alone using simple nearest neighbor thermodynamic models (3). In contrast, tertiary structure formation is a subtle competition between intrinsic flexibility of single-stranded segments, rigidity imparted from base-stacking interactions, stabilization of noncanonical hydrogen bonding patterns, and site-specific ion binding. In principle, a molecular dynamics simulation using a properly calibrated force field should capture all of the physicochemical properties of ribonucleotides relevant to the RNA folding process. Up until now, however, even small, fast-folding tetraloops cannot be accurately and reversibly folded from the unfolded state (4-6). In contrast, numerous documented successes have been reported using de novo protein folding with all-ato...

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Section: Discussionmentioning

confidence: 99%

High-resolution reversible folding of hyperstable RNA tetraloops using molecular dynamics simulations

Chen

Garcı́a

2013

Proc. Natl. Acad. Sci. U.S.A.

284

497

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“…This was shown by the lack of replication of the ΔU41C42/U47C48 mutant which contains a regular RNA stem with a GAUA tetraloop that is further stabilized by the closing C-G base pair [15], [21], [22]. The ΔU41C42/U47C48 mutant did not support viral RNA replication.…”

Section: Discussionmentioning

confidence: 99%

Unusual Loop-Sequence Flexibility of the Proximal RNA Replication Element in EMCV

et al. 2011

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Picornaviruses contain stable RNA structures at the 5′ and 3′ ends of the RNA genome, OriL and OriR involved in viral RNA replication. The OriL RNA element found at the 5′ end of the enterovirus genome folds into a cloverleaf-like configuration. In vivo SELEX experiments revealed that functioning of the poliovirus cloverleaf depends on a specific structure in this RNA element. Little is known about the OriL of cardioviruses. Here, we investigated structural aspects and requirements of the apical loop of proximal stem-loop SL-A of mengovirus, a strain of EMCV. Using NMR spectroscopy, we showed that the mengovirus SL-A apical loop consists of an octaloop. In vivo SELEX experiments demonstrated that a large number of random sequences are tolerated in the apical octaloop that support virus replication. Mutants in which the SL-A loop size and the length of the upper part of the stem were varied showed that both stem-length and stability of the octaloop are important determinants for viral RNA replication and virus reproduction. Together, these data show that stem-loop A plays an important role in virus replication. The high degree of sequence flexibility and the lack of selective pressure on the octaloop argue against a role in sequence specific RNA-protein or RNA-RNA interactions in which octaloop nucleotides are involved.

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“…It is interesting to note that a closing base pair of C-G occurs as frequently as all five of the remaining base pairs combined (see Table 1, data set 3). Recently, Blose et al (2009) have investigated the molecular basis for the enhanced stability of tetraloops with C-G closing base pairs. Although they may be more stable, it is unclear why C-G closing base pairs are so common in nature, as nature does not select tetraloop sequences based solely on stability (see Table 2).…”

Section: Thermodynamics Of Rna Tetraloopsmentioning

confidence: 99%

Thermodynamic characterization of naturally occurring RNA tetraloops

Sheehy

Davis²,

Znosko³

2010

RNA

112

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Although tetraloops are one of the most frequently occurring secondary structure motifs in RNA, less than one-third of the 30 most frequently occurring RNA tetraloops have been thermodynamically characterized. Therefore, 24 stem-loop sequences containing common tetraloops were optically melted, and the thermodynamic parameters DH°, DS°, DG°3 7, and T M for each stem-loop were determined. These new experimental values, on average, are 0.7 kcal/mol different from the values predicted for these tetraloops using the model proposed by Vecenie CJ, Morrow CV, Zyra A, Serra MJ. 2006. Biochemistry 45: 1400-1407. The data for the 24 tetraloops reported here were then combined with the data for 28 tetraloops that were published previously. A new model, independent of terminal mismatch data, was derived to predict the free energy contribution of previously unmeasured tetraloops. The average absolute difference between the measured values and the values predicted using this proposed model is 0.4 kcal/mol. This new experimental data and updated predictive model allow for more accurate calculations of the free energy of RNA stem-loops containing tetraloops and, furthermore, should allow for improved prediction of secondary structure from sequence. It was also shown that tetraloops within the sequence 59-GCCNNNNGGC-39 are, on average, 0.6 kcal/mol more stable than the same tetraloop within the sequence 59-GGCNNNNGCC-39. More systemic studies are required to determine the full extent of non-nearest-neighbor effects on tetraloop stability.

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Contribution of the Closing Base Pair to Exceptional Stability in RNA Tetraloops: Roles for Molecular Mimicry and Electrostatic Factors

Cited by 39 publications

References 63 publications

High-resolution reversible folding of hyperstable RNA tetraloops using molecular dynamics simulations

High-resolution reversible folding of hyperstable RNA tetraloops using molecular dynamics simulations

Unusual Loop-Sequence Flexibility of the Proximal RNA Replication Element in EMCV

Thermodynamic characterization of naturally occurring RNA tetraloops

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