RNA folding causes secondary structure rearrangement

Abstract

The secondary structure of the P5abc subdomain (a 56-nt RNA) of the Tetrahymena thermophila group I intron ribozyme has been determined by NMR. Its base pairing in aqueous solution in the absence of magnesium ions is significantly different from the RNA in a crystal but is consistent with thermodynamic predictions. On addition of magnesium ions, the RNA folds into a tertiary structure with greatly changed base pairing consistent with the crystal structure: three Watson-Crick base pairs, three G⅐U base pairs, and an extra-stable tetraloop are lost. The common assumption that RNA folds by first forming secondary structure and then forming tertiary interactions from the unpaired bases is not always correct.RNA plays many roles in biological processes, and our knowledge of its importance is still expanding rapidly (1, 2). By understanding how RNA folds into its three-dimensional structure, we can begin to predict its folding from its sequence and can greatly improve our understanding of its biological functions. The standard RNA folding mechanism is a two-step process. The RNA first folds into a secondary structure, which then folds into a three-dimensional tertiary structure stabilized by interactions between the preformed secondary structural motifs (3-7). Divalent metal ions bound at a few specific locations appear to be crucial for the tertiary folding (8, 9). RNA secondary structures can be deduced from a phylogenetic comparison of many related sequences or from a calculation of free energies (based on thermodynamic measurements of model oligonucleotides) for stable secondary structures. Secondary structures and low-resolution tertiary structures can also be obtained from biochemical experiments that reveal nucleotide accessibility and detect long-range interactions. However, NMR spectroscopy and x-ray diffraction are the major tools for high-resolution structure determination of RNA.The crystal structure of the P4-P6 domain of the Tetrahymena group I intron at high resolution (10-12) has revealed a tremendous amount of new information on RNA structure and folding. When the Tetrahymena group I intron (414 nt) folds into a catalytically active form, the 160-nt P4-P6 domain is the first to fold (6,13,14). Cleavage experiments with Fe-EDTA showed that the isolated domain and the domain in the intron fold into the same tertiary structure on addition of millimolar concentrations of magnesium ions (15). A cluster of five Mg 2ϩ were found coordinated to phosphate oxygen atoms in the A-rich bulge and in the 3-helix junction of the P5abc subdomain of the P4-P6 domain (10, 11). To confirm the identities of the metal-coordinated phosphate groups, phosphorothioate substitution experiments were performed on the 160-nt P4-P6 domain and a 56-nt P5abc subdomain (9). Native gel electrophoresis experiments separated two forms of the RNA molecules: folded and unfolded. By systematically replacing sulfur atoms for oxygen atoms, seven pro-Rp phosphate oxygens were found to be necessary for tertiary folding in solution in bo...

Maximizing RNA folding rates: A balancing act

“…The arguments presented in this article also highlight the fact that secondary and tertiary interactions must be compatible for efficient assembly of independent folding domains, as illustrated by the coupling of secondary and tertiary folding in the P5abc RNA and a complex pseudoknot in the Escherichia coli a operon (Gluick & Draper, 1994;Wu & Tinoco, 1998)+ As the helices become increasing stable and rigid, rearrangement of the secondary structure becomes less probable and may impede the formation of tertiary interactions+ Thus, optimal folding rates require a balance between secondary and tertiary interactions, an idea that was proposed some time ago by Go (1983) in the context of protein folding+ FIGURE 3. Relationship between folding times (t) and relative domain stabilities (⌬G 46 /⌬G 39 )+ On the right side of the graph, the minimum kinetic barrier ⌬G I ‡ is proportional to the stability of the P4-P6 domain, ⌬G 46 + This corresponds to the folding kinetics of the wildtype ribozyme+ On the left, the minimum barrier depends on the free energy for folding the P3-P9 domain, ⌬G 39 + The shortest folding times are predicted to occur when these trends converge (⌬G 46 / ⌬G 39 ; 1)+ The complete curve can be approximated by the sum of the terms in equation (3)+ Many group I ribozymes (and other RNAs) contain structural elaborations that reinforce the central fold (Michel & Westhof, 1990;Westhof et al+, 1996)+ These peripheral "stability elements," such as the P5abc threehelix junction, appear to lengthen the folding time of the Tetrahymena ribozyme Russell & Herschlag, 1999)+ This differs from the idea that a stable nugget of tertiary structure, which in this case is outside of the structural core, functions as a scaffold upon which the rest of the structure is built+ Interestingly, the stabilizing function of the P5abc region is replaced by the protein CYT-18 in group I introns from Neurospora mitochondria (Mohr et al+, 1992;Caprara et al+, 1996)+ This hints at the possibility that RNA-protein complexes offer the advantage of a more flexible assembly process, in which the RNA-RNA interactions remain fluid until they are solidified by binding of proteins (e+g+, see Buchmueller et al+, 2000)+…”

“…Three-state folding mechanism+ FIGURE 2. Kinetic partitioning mechanism for folding of the Tetrahymena ribozyme+ The unfolded (U) RNA is represented by an ensemble of structures in the absence of MgCl 2 + The majority of the population folds via intermediates (I) that are stabilized by native interactions in the P4-P6 domain (green cylinders) and misfolded structures in P3 (yellow)+ The slow transition from I to the native structure (N) requires partial or complete unfolding of the RNA, corresponding to a large transition state energy (⌬G I ‡ )+ A small fraction of the wild-type RNA folds rapidly+ Stabilization of P3 by the mutation U273 to A enables ;80% of the RNA to form N without populating the I states (right)+ Mutations that change the energy levels of I and N may also change the position of the transition state, which we do not indicate here for simplicity+ The transition state itself is a collection of related structures+ quires tertiary interactions involving nucleotides that are far apart in the sequence+ This distinction between local and long-range interactions is captured by hierarchical models of RNA structure (Brion & Westhof, 1997)+ In general, there is a greater propensity to form local structures because these are entropically favorable+ An example of this is rapid folding of the P5abc subdomain of the Tetrahymena ribozyme in the absence of long-range interactions with P2 or with the P3-P9 domain+ Local structure is more likely to be stable in RNA than in proteins because the base pairs that define RNA secondary structure are strong relative to tertiary interactions (Banerjee et al+, 1993;Herschlag, 1995;Draper, 1996)+ The seemingly opposing demands of forming local structure and establishing long-range interactions that define global conformation results in "topological frustration" (Thirumalai & Woodson, 1996)+ This term, borrowed from condensed matter physics, expresses the inability of individual residues to simultaneously satisfy all energetically favorable interactions with their neighbors (Toulouse, 1977)+ One example of topological frustration is the competition between two base pairings of the P5c helix, only one of which is compatible with native tertiary interactions in the P5abc subdomain (Wu & Tinoco, 1998)+ Another example is misfolding of P3, in which the two strands joining P7 and P8 base pair with each other instead of pairing with nucleotides 170 residues upstream to form the native P3 pseudoknot (Pan & Woodson, 1998)+ The incorrect pairing is kinetically preferred over P3, at least in part because it involves short-range rather than long-range contacts+ If local interactions in one part of the ribozyme dominate the energetic landscape (such as P5abc or mispaired P3), then their premature formation stabilizes intermediate structures and slows down the overall folding rate+ Conversely, if interactions throughout the RNA have roughly similar energetics, then the native structure is more likely to form cooperatively and rapidly, with few or no apparent intermediates+…”

RNA structural motifs: building blocks of a modular biomolecule

“…Large structural rearrangements have been observed on metal binding (Wu & Tinoco, 1998 ;Penedo et al 2004) indicating that secondary structure is not always fixed prior to formation of tertiary interactions.…”

A simple and efficient method to reduce nontemplated nucleotide addition at the 3′ terminus of RNAs transcribed by T7 RNA polymerase

“…Four additional DNA templates that should direct the synthesis of RNAs from 27 to 64 nt were examined+ The modified DNAs were named P1, V⌿, M5-54, and M5-64, and their 59 terminal nucleotides were, respectively, 59GA39, 59AT39, 59GG39, and 59GG39+ The transcription products from each pair of unmodified and modified DNAs are shown in Figure 2A+ The unmodified versions of all four DNAs generated significant amounts of the respective Nϩ1 products (Fig+ 2A, lanes 1, 4, 5, and 7) whereas the modified DNAs did not (Fig+ 2, lanes 2, 3, 6, and 8)+ We have now produced four additional RNAs from modified templates that had significantly reduced Nϩ1 RNAs+ Transcription reactions using P1 and V⌿ generated RNAs that were either 1 or 2 nt less than full-length+ The modified DNA templates did not significantly affect the abundance of these truncated RNAs (Fig+ 2A, lanes 1-4)+ Transcripts longer than 50 nt are difficult to purify away from their associated Nϩ1 RNAs even after extensive preparative gel electrophoresis+ This has led to contamination of the desired transcripts that can complicate spectroscopic and biochemical analyses of RNAs+ For example, Wu and Tinoco (1998) observed in a 56-nt RNA three additional imino signals associated with the last three base pairs of the Nϩ1 RNA+ In one-dimensional imino spectrum of the 56-nt RNA (Fig+ 2B, bottom spectrum), the peak at 12+58 ppm (labeled G191 ϩ ) and the shoulder at 13+2 ppm (labeled G131 ϩ ) were tentatively assigned to the Nϩ1 RNA because it was not possible to separate the Nϩ1 RNA from the 56-nt RNA+ To examine whether transcripts purified from the modified template could remove the confounding signals, we purified the transcripts from a modified template encoding the 56-nt sequence used by Wu and Tinoco and performed one-dimensional imino spectra analysis (Fig+ 2B, top spectrum)+ The transcript from the modified template, M5-56**, lacked the G191 ϩ signal, was significantly enhanced in the signal for G131 and G191, and did not have the extra peak shoulder associated with G131 ϩ + All other peaks were unaffected, demonstrating that the nucleotide sequence produced from modified template M5-56 was identical to the expected sequence+ FIGURE 2. Effect of ribose 29-methoxy modifications at the last two nucleotides at the 59 termini of four DNA templates on Nϩ1 activity of T7 RNA polymerase+ A: RNAs in denaturing polyacylamide gels stained with Toluidine Blue+ Both gels contain 20% polyacrylamide, but the samples in lanes 1-4 and lanes 5-6 were electrophoresed for 16 and 24 h, respectively+ The DNA templates used to generate the transcripts are listed on top of the gel images, with two asterisks denoting modification of the 59 terminal two nucleotides with ribose 29-methoxyl moieties+ The lengths of the transcripts expected for P1 and V⌿ are indicated on the left of the gel image whereas transcripts from M5-56 and M5-64 are shown on the right side+ The positions of the RNA 1 nt longer than the expected size are indicated with "Nϩ1"+ B: NMR spectra of the imino protons of the RNAs transcribed from M5-56 and M5-56**+ The positions of G131 and G191 are identified within the spectra as assigned by Wu and Tinoco (1998)+ G131 ϩ and G191 ϩ are the peaks associated with an RNA that contains the Nϩ1 nucleotide+…”

“…Effect of ribose 29-methoxy modifications at the last two nucleotides at the 59 termini of four DNA templates on Nϩ1 activity of T7 RNA polymerase+ A: RNAs in denaturing polyacylamide gels stained with Toluidine Blue+ Both gels contain 20% polyacrylamide, but the samples in lanes 1-4 and lanes 5-6 were electrophoresed for 16 and 24 h, respectively+ The DNA templates used to generate the transcripts are listed on top of the gel images, with two asterisks denoting modification of the 59 terminal two nucleotides with ribose 29-methoxyl moieties+ The lengths of the transcripts expected for P1 and V⌿ are indicated on the left of the gel image whereas transcripts from M5-56 and M5-64 are shown on the right side+ The positions of the RNA 1 nt longer than the expected size are indicated with "Nϩ1"+ B: NMR spectra of the imino protons of the RNAs transcribed from M5-56 and M5-56**+ The positions of G131 and G191 are identified within the spectra as assigned by Wu and Tinoco (1998)+ G131 ϩ and G191 ϩ are the peaks associated with an RNA that contains the Nϩ1 nucleotide+…”