Relative Stability of Helices Determines the Folding Landscape of Adenine Riboswitch Aptamers

Lin, Jong-Chin; Thirumalai, D.

doi:10.1021/ja8063638

Cited by 84 publications

(121 citation statements)

References 18 publications

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“…Although the coarse-grained (CG) models have a number of limitations, they have been proved useful in deciphering the key features that control the folding of a variety of RNA molecules (27,(37)(38)(39)(40). Comparisons of our simulations of RNA pseudoknots to previously observed equilibrium optical and calorimetric melting profiles show excellent agreement.…”

Section: Discussionsupporting

confidence: 53%

Assembly mechanisms of RNA pseudoknots are determined by the stabilities of constituent secondary structures

Cho

Pincus

Thirumalai

2009

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

Self Cite

109

156

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Understanding how RNA molecules navigate their rugged folding landscapes holds the key to describing their roles in a variety of cellular functions. To dissect RNA folding at the molecular level, we performed simulations of three pseudoknots (MMTV and SRV-1 from viral genomes and the hTR pseudoknot from human telomerase) using coarse-grained models. The melting temperatures from the specific heat profiles are in good agreement with the available experimental data for MMTV and hTR. The equilibrium free energy profiles, which predict the structural transitions that occur at each melting temperature, are used to propose that the relative stabilities of the isolated helices control their folding mechanisms. Kinetic simulations, which corroborate the inferences drawn from the free energy profiles, show that MMTV folds by a hierarchical mechanism with parallel paths, i.e., formation of one of the helices nucleates the assembly of the rest of the structure. The SRV-1 pseudoknot, which folds in a highly cooperative manner, assembles in a single step in which the preformed helices coalesce nearly simultaneously to form the tertiary structure. Folding occurs by multiple pathways in the hTR pseudoknot, the isolated structural elements of which have similar stabilities. In one of the paths, tertiary interactions are established before the formation of the secondary structures. Our work shows that there are significant sequence-dependent variations in the folding landscapes of RNA molecules with similar fold. We also establish that assembly mechanisms can be predicted using the stabilities of the isolated secondary structures.kinetic partitioning mechanism ͉ parallel pathways ͉ ribosomal frameshifting ͉ RNA folding T he RNA folding problem has taken center stage in molecular biology because these molecules play a vital role in a variety of cellular functions. The percentage of the transcribed noncoding sequences in mice and human genomes exceed 90% (1), and the functional importance of the rest of the noncoding RNA is now only beginning to be understood (2). The noncoding roles of ribosomal RNA (rRNA) and transfer RNA (tRNA) are well known, but the discovery of ribozymes (RNA enzymes) has sparked an intense search for the functional roles of the rest of the noncoding RNA molecules (2, 3), which include catalysis, replication, transcriptional and translational regulation, and ligand binding (4), just to name a few. Consequently, it is important to determine the mechanisms by which RNA molecules fold so that a deeper understanding of the structure-function relationship can emerge.Although great progress has been made in understanding of how RNA molecules fold (5Ϫ10) global principles that determine the sequence dependent folding mechanisms have not fully emerged. It is argued that RNA folding mechanisms must be inherently simple because of the apparent separation in the energy scales that describe the different levels of structural organization (11). Furthermore, RNA molecules are constructed from only four nucleotides (nts), ...

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Section: Discussionsupporting

confidence: 53%

Assembly mechanisms of RNA pseudoknots are determined by the stabilities of constituent secondary structures

Cho

Pincus

Thirumalai

2009

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

Self Cite

109

156

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“…The observed folding landscape highlighted that P3 folded at a higher force than P2 in the add A-riboswitch (different from what Greenleaf et al previously observed for pbuE aptamer). This was already predicted by a Langevin dynamics study of the add A-riboswitch (Lin and Thirumalai 2008), and it may be due to the different stability of the helices P2 and P3 in the two aptamers. The authors observed also that P1 in the add aptamer is similar in stability to P2 and P3 even in the absence of the cognate ligand, adenine.…”

Section: Introductionmentioning

confidence: 59%

“…In their single-molecule force measurement experiment of the pbuE A-riboswitch, Greenleaf et al (2008) saw that P3 unfolds before P2, indicating less stability of P3. In Langevin simulations of the aptamer domain of the add A-riboswitch (Lin and Thirumalai 2008), however, P2 was found to unfold before P3. This agrees with recent results from single-molecule experiments performed by Neupane et al (2011) on the add A-riboswitch.…”

Section: Discussionmentioning

confidence: 96%

Loop–loop interaction in an adenine-sensing riboswitch: A molecular dynamics study

Allnér

Nilsson

Villa

2013

RNA

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Riboswitches are mRNA-based molecules capable of controlling the expression of genes. They undergo conformational changes upon ligand binding, and as a result, they inhibit or promote the expression of the associated gene. The close connection between structural rearrangement and function makes a detailed knowledge of the molecular interactions an important step to understand the riboswitch mechanism and efficiency. We have performed all-atom molecular dynamics simulations of the adenine-sensing add A-riboswitch to study the breaking of the kissing loop, one key tertiary element in the aptamer structure. We investigated the aptamer domain of the add A-riboswitch in complex with its cognate ligand and in the absence of the ligand. The opening of the hairpins was simulated using umbrella sampling using the distance between two loops as the reaction coordinate. A two-step process was observed in all the simulated systems. First, a general loss of stacking and hydrogen bond interactions is seen. The last interactions that break are the two base pairs G37-C61 and G38-C60, but the break does not affect the energy profile, indicating their pivotal role in the tertiary structure formation but not in the structure stabilization. The junction area is partially organized before the kissing loop formation and residue A24 anchors together the loop helices. Moreover, when the distance between the loops is increased, one of the hairpins showed more flexibility by changing its orientation in the structure, while the other conserved its coaxial arrangement with the rest of the structure.

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“…2B). We rationalize these observations using a kinetic partitioning mechanism (30,31), whereby increasing P2 stem strength increases the population of correctly folded riboswitches available to perform gene regulation, by minimizing the occurrence of the misfolded mRNA transcripts. As a consequence of increasing the P2 stem strength within the M6′ and M6′′ mutants, there also appears to be a change in the relative order of helix stability (Table S1).…”

Section: Mutations Within the P2 Stem Increase Absolute Reporter Genementioning

confidence: 91%

Reengineering orthogonally selective riboswitches

Dixon

Duncan²,

Geerlings³

et al. 2010

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

151

163

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The ability to independently control the expression of multiple genes by addition of distinct small-molecule modulators has many applications from synthetic biology, functional genomics, pharmaceutical target validation, through to gene therapy. Riboswitches are relatively simple, small-molecule-dependent, protein-free, mRNA genetic switches that are attractive targets for reengineering in this context. Using a combination of chemical genetics and genetic selection, we have developed riboswitches that are selective for synthetic "nonnatural" small molecules and no longer respond to the natural intracellular ligands. The orthogonal selectivity of the riboswitches is also demonstrated in vitro using isothermal titration calorimetry and x-ray crystallography. The riboswitches allow highly responsive, dose-dependent, orthogonally selective, and dynamic control of gene expression in vivo. It is possible that this approach may be further developed to reengineer other natural riboswitches for application as small-molecule responsive genetic switches in both prokaryotes and eukaryotes.

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Relative Stability of Helices Determines the Folding Landscape of Adenine Riboswitch Aptamers

Cited by 84 publications

References 18 publications

Assembly mechanisms of RNA pseudoknots are determined by the stabilities of constituent secondary structures

Assembly mechanisms of RNA pseudoknots are determined by the stabilities of constituent secondary structures

Loop–loop interaction in an adenine-sensing riboswitch: A molecular dynamics study

Reengineering orthogonally selective riboswitches

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