Metal binding and base ionization in the U6 RNA intramolecular stem-loop structure

Huppler, Anna R.; Nikstad, L.J.; Allmann, A.M.; Brow, David A.; Butcher, Samuel E.

doi:10.1038/nsb800

Cited by 147 publications

(226 citation statements)

References 25 publications

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“…Magnesium and potassium, the dominant counterions for RNA folding in vivo, are unfortunately spectroscopically invisible. NMR chemical shift perturbation mapping is a sensitive, but indirect, means of detecting ion association with RNA (Butcher et al 2000;Huppler et al 2002;Sigel et al 2004;Fan et al 2005), and can be difficult to interpret if ion binding simultaneously induces conformational change. Direct methods for detecting sites of metal ion association include Nuclear Overhauser effects (NOEs) to cobalt hexammine, and manganese-induced paramagnetic relaxation enhancement (PRE) (Bertini and Luchinat 1986;Kieft and Tinoco 1997;Gonzalez and Tinoco 1999;Butcher et al 2000).…”

Section: Introductionmentioning

confidence: 99%

Role of metal ions in the tetraloop–receptor complex as analyzed by NMR

Davis

Foster²,

Tonelli³

et al. 2006

RNA

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Metal ions are critical for the proper folding of RNA, and the GAAA tetraloop-receptor is necessary for the optimal folding and function of many RNAs. We have used NMR to investigate the role of metal ions in the structure of the tetraloop-receptor in solution. The NMR data indicate native tertiary structure is formed under a wide range of ionic conditions. The lack of conformational adaptation in response to very different ionic conditions argues against a structural role for divalent ions. Nuclear Overhauser effects to cobalt hexammine and paramagnetic relaxation enhancement induced by manganese ions were used to determine the NMR structures of the tetraloop receptor in association with metal ions, providing the first atomic-level view of these interactions in the solution state. Five manganese and two cobalt hexammine ions could be localized to the RNA surface. The locations of the associated metal ions are similar, but not identical to, those of previously determined crystal structures. The sites of association are in general agreement with nonlinear Poisson-Boltzmann calculations of the electrostatic surface, emphasizing the general importance of diffusely associated ions in RNA tertiary structure.

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Section: Introductionmentioning

confidence: 99%

Role of metal ions in the tetraloop–receptor complex as analyzed by NMR

Davis

Foster²,

Tonelli³

et al. 2006

RNA

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“…We find that the U80G ISL displays stereospecific chemical shift changes at G80 upon cadmium addition that are qualitatively similar to those of the wild-type ISL ( Figure 6A). Cadmium binding to phosphorothioates typically produces a diagnostic upfield change in the 31 P chemical shift, as observed for the S p resonance 5′ to nucleotide 80 for both the wild-type and mutant ISL RNAs ( Figure 6A) (12,33,34). As previously observed for the wild-type U6 ISL, these data cannot be fit to a single-binding site model, because cadmium ions also bind nonspecifically to the electronegative major groove of the ISL, and sample aggregation and line broadening occur with higher molar equivalents of cadmium (12).…”

Section: Metal Binding Studiesmentioning

confidence: 69%

“…The phosphorothioate-substituted RNA was purified by denaturing PAGE, anion exchange, and gel filtration chromatography as described above. Cadmium binding was monitored by titrating cadmium chloride into the sample and by following the chemical shift changes in the S p and R p phosphorothioate signals by 31 P 1D NMR at 202 MHz (phosphorus frequency), as previously described (12).…”

Section: Metal Binding Studiesmentioning

confidence: 99%

“…Thirty-three residual dipolar couplings were measured and used to improve the long-range order of the structure. RDCs were not included for regions of the pentaloop structure that are suspected to be dynamic from previous structure calculations (12). The axial component for the principal alignment tensor (D a ) and rhombicity (R) was determined from a powder-pattern analysis and adjusted by a grid search (23).…”

Section: Structure Calculations With Residual Dipolar Couplingsmentioning

confidence: 99%

“…Using these methods, we have determined the structure of the U6 ISL harboring the lethal U80G mutation. The mutation leads to the formation of a new Watson-Crick base pair in the internal loop, between G80 and C67, which in turn disrupts the phylogenetically conserved C67·A79 wobble pair that forms in the wild-type structure (12) (Figure 1). Thermodynamic studies reveal that this altered secondary structure causes an increase in stability (ΔΔG fold = −3.6 ± 1.9 kcal/mol) over that of the wild-type ISL.…”

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Structural Basis for a Lethal Mutation in U6 RNA^,

et al. 2003

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U6 RNA is essential for nuclear pre-mRNA splicing and has been implicated directly in catalysis of intron removal. The U80G mutation at the essential magnesium binding site of the U6 3′ intramolecular stem-loop region (ISL) is lethal in yeast. To further understand the structure and function of the U6 ISL, we have investigated the structural basis for the lethal U80G mutation by NMR and optical spectroscopy. The NMR structure reveals that the U80G mutation causes a structural rearrangement within the ISL resulting in the formation of a new Watson-Crick base pair (C67·G80), and disrupts a protonated C67·A79 wobble pair that forms in the wild-type structure. Despite the structural change, the accessibility of the metal binding site is unperturbed, and cadmium titration produces similar phosphorus chemical shift changes for both the U80G mutant and wild-type RNAs. The thermodynamic stability of the U80G mutant is significantly increased (ΔΔG fold = −3.6 ± 1.9 kcal/mol), consistent with formation of the Watson-Crick pair. Our structural and thermodynamic data, in combination with previous genetic data, suggest that the lethal basis for the U80G mutation is stem-loop hyperstabilization. This hyperstabilization may prevent the U6 ISL melting and rearrangement necessary for association with U4.Proteomic diversity in eukaryotes is generated by alternative splicing of exons from nuclear premessenger RNA (pre-mRNA) by the spliceosome. The spliceosome is a large ribonucleoprotein complex, made up of five small nuclear RNAs (snRNAs), U1, U2, and U4, U5, U6, and more than 70 proteins (1-4). The spliceosome catalyzes a two-step transesterification reaction, speculated to be RNA-catalyzed by analogy to mitochondrial group II self-splicing ribozymes (2,3,5). Two snRNAs (U2 and U6) likely comprise part of the spliceosome active site, and U6 is essential for catalysis (2,3,5). In the active spliceosome, U2 and U6 form a complex by base pairing to each other, and have also been shown to base pair to pre-mRNA at the first step of splicing. Moreover, mutagenesis data have shown that certain regions of U6 must be intact for the assembled spliceosome to catalyze the first or second step of splicing (6). Other atomic substitution studies of U6 RNA have revealed several phosphate oxygens that are essential for splicing (7-9), which are potential sites of magnesium coordination required by the spliceosome. Recently, the U2-U6 complex was shown to catalyze a reaction similar to the first step of splicing in the † This work was supported by NIH Grant GM65166. S.R.V. absence of protein, lending more evidence to the hypothesis of an RNA active site in the spliceosome (10).The formation of the U2-U6 complex is initiated by a large conformational change in U6 RNA (1, 11). During spliceosome assembly, U6 extensively base pairs with U4 RNA, forming a helical secondary structure. After the spliceosome is assembled completely, base pairing between U4 and U6 is disrupted and U6 undergoes a large conformational change, in which an intramolec...

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RNA Structure: Tetraloops

Cheong

Kim

Cheong

2015

Encyclopedia of Life Sciences

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RNA hairpins are among the most common RNA secondary structural elements and are frequently capped by RNA tetraloops. RNA tetraloops are composed of characteristic four‐loop nucleotides that form a compact and stable structure. While they can be formed by many different nucleotide sequences, UNCG (N = A, C, G, or U), GNRA (R = A or G), and CUUG tetraloops are found most often. Tetraloops usually help initiate RNA ‐folding processes and provide sites for tertiary contacts within or between RNAs and for protein binding, thereby facilitating the assembly of ribonucleoprotein particles. Tetraloop interactions can be either sequence‐ or structure‐specific. Herein, we discuss the structures of RNA tetraloops and their interactions with other RNA structural motifs and with RNA ‐binding proteins. Key Concepts RNA hairpins play important structural and functional roles in RNA. RNA tetraloops are composed of four‐loop nucleotides that form a compact and stable structure. Tetraloops assist RNA folding and provide sites for RNA–RNA and RNA–protein interactions. Tetraloop interactions can be either sequence‐ or structure‐specific. We discuss the structures of RNA tetraloops and their interactions with other RNAs and proteins.

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Metal binding and base ionization in the U6 RNA intramolecular stem-loop structure

Cited by 147 publications

References 25 publications

Role of metal ions in the tetraloop–receptor complex as analyzed by NMR

Role of metal ions in the tetraloop–receptor complex as analyzed by NMR

Structural Basis for a Lethal Mutation in U6 RNA^,

RNA Structure: Tetraloops

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Metal binding and base ionization in the U6 RNA intramolecular stem-loop structure

Cited by 147 publications

References 25 publications

Role of metal ions in the tetraloop–receptor complex as analyzed by NMR

Role of metal ions in the tetraloop–receptor complex as analyzed by NMR

Structural Basis for a Lethal Mutation in U6 RNA,

RNA Structure: Tetraloops

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Structural Basis for a Lethal Mutation in U6 RNA^,