Divalent metal ions promote the formation of the 5′-splice site recognition complex in a self-splicing group II intron

Kruschel, Daniela; Sigel, Roland K. O.

doi:10.1016/j.jinorgbio.2008.08.006

Cited by 20 publications

(20 citation statements)

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“…Additionally, NMR is applied to characterize site-specific M 2+ binding. In the d3′EBS1*/IBS1* sequence pair used herein, two AUs are replaced by GCs for proper duplex formation, a modification that does not affect the first transesterification step (25,26). For FRET experiments, the 5′-ends of the d3′EBS1* hairpin and the seven-nucleotide-long IBS1* are labeled with Cy3 and Cy5, respectively (Fig.…”

Section: +mentioning

confidence: 99%

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Cation-induced kinetic heterogeneity of the intron–exon recognition in single group II introns

Kowerko

König

Skilandat

et al. 2015

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

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RNA is commonly believed to undergo a number of sequential folding steps before reaching its functional fold, i.e., the global minimum in the free energy landscape. However, there is accumulating evidence that several functional conformations are often in coexistence, corresponding to multiple (local) minima in the folding landscape. Here we use the 5′-exon-intron recognition duplex of a self-splicing ribozyme as a model system to study the influence of Mg 2+ NA folding is a hierarchical process that depends on the sequential formation of secondary and tertiary structures. As the RNA phosphate-sugar backbone is negatively charged, structural compaction creates electrostatic repulsion, which must be overcome by positive charges. The majority of negative charges are nonspecifically screened by the ion atmosphere, typically a set of dynamically exchanging M + ions (1). An estimated 10-20% of negative charge is, however, compensated by M n+ that bind site-specifically to the RNA molecule, in particular, Mg 2+ (2). One RNA molecule that is known to harbor several specific M 2+ binding sites is the self-splicing group II intron Sc.ai5γ from the yeast mitochondrial cox1 (cytochrome oxidase 1) gene (3). It is one of the largest known RNA enzymes, and both its folding pathway and catalysis are strictly dependent on Mg 2+. In turn, the splicing reaction is inhibited by small amounts of Ca 2+ (4). Site specificity of the two sequential transesterfication reactions is ensured by proper base pairing between distal exon-binding sites (5′ cleavage, EBS1 and 2; 3′ cleavage, EBS3) and intron-binding sites (IBS1, 2, and 3) (5).Single-molecule Förster resonance energy transfer (smFRET), i.e., distance-dependent energy transfer between a single pair of fluorophores, is ideally suited to study the cation-dependent conformational dynamics of single RNA molecules (6, 7). If different conformations lead to distinctly different transfer efficiencies, smFRET unveils the entire folding pathway, reports on the relative occurrence of all conformations present in the ensemble, and provides detailed information on the rates at which they interconvert (7). This is important because simple two-state folding is rarely observed in experimental data (8, 9). Rather, the vast conformational space sampled by biomolecules often results not only in folding intermediates but also in kinetic traps and/or multiple native states. In an smFRET experiment, individual molecules consequently display different behaviors that may or may not persist over the observation period (10). Heterogeneity has been precedented for a number of RNA molecules, including group I introns (11, 12), the hairpin ribozyme (13-17), and RNase P RNA (18). In addition, heterogeneity has been reported for different . However, the molecular basis of the phenomenon is often enigmatic, and its quantitative characterization is challenging (21).Here we use the 5′-exon-intron recognition site of the Sc.ai5γ ribozyme to study Mg 2+ -and Ca 2+ -mediated RNA-RNA structure formation by smFRET. ...

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Section: +mentioning

confidence: 99%

“…concentration (25). Native PAGE with the Cy3-d3′EBS1*/Cy5-IBS1* system shows that (i) the attached fluorophores do not hamper strand association and (ii) M 2+ further promotes their interaction (SI Appendix, Fig.…”

Section: +mentioning

confidence: 99%

Cation-induced kinetic heterogeneity of the intron–exon recognition in single group II introns

Kowerko

König

Skilandat

et al. 2015

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

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“…B, d3ЈEBS1⅐dIBS1, the RNA⅐DNA hybrid construct used in this study. The sequences of nucleotides 5-25 of the RNA (d3ЈEBS1) containing EBS1 (purple) and of dIBS1 DNA (green) are derived from the Sc.ai5␥ intron found in the cox1 gene of S. cerevisiae mitochondria The base pairs marked with light green/dark purple letters were mutated from A⅐T to G⅐C for the sake of stability (57). The nucleotides 1-4 and 26 -29 (boxed) are added to the wild type sequence.…”

Section: Methodsmentioning

confidence: 99%

The Role of Mg(II) in DNA Cleavage Site Recognition in Group II Intron Ribozymes

Skilandat

Sigel

2014

Journal of Biological Chemistry

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Background: Group II introns cleave DNA and RNA 3Ј of a short duplex formed between the intron and the target. Results: We present the NMR structure of this hybrid duplex and describe two distinct Mg 2ϩ binding sites. Conclusion:The hybrid is asymmetric and strongly stabilized by Mg 2ϩ binding. Significance: Site-bound metal ions are crucially important for group II intron cleavage site recognition.

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“…1). [15][16][17][18][19][20][21][22] One of the biggest challenges is the determination of the metal ion affinities because usually several metal ions simultaneously bind to one RNA molecule with similar affinities. We have thus developed an iterative approach to calculate the intrinsic binding constants of different metal ions to one ligand, e.g.…”

Section: Metal Ions In the Catalytic Center Of Group II Intron Ribozymesmentioning

confidence: 99%

Shaping RNA Structures with Metal Ions and Metal Ion Complexes

Sigel

Gallo²

2010

Chimia

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The research in our laboratory focuses on the role of metal ions and their complexes in structure formation and folding of nucleic acids. Large catalytic RNAs, like group II introns and some riboswitches, as well as shorter RNAs and DNAs containing modified nucleotides for the assembly of nanodevices are examined. Abundant metal ions like Mg2+ or natural metabolites like coenzyme B-12 are in the center of interest, but also other metal ions, complexes thereof and B-12 derivatives are applied with the aim to understand the largely unknown and manifold non-covalent interactions with nucleic acids. We apply a multitude of techniques, including potentiometric pH titrations, NMR spectroscopy, X-ray crystallography, gel electrophoresis and single molecule FRET experiments. Here we briefly summarize each of our research topics emphasizing the interaction of coenzyme B-12 and its derivatives with the btuB riboswitch of E. coli. This highly conserved sequence, found in the 5'-untranslated region (5'-UTR) of the btuB mRNA, is involved in the regulation of the btuB protein expression. After a summary on the historical discovery of such riboswitches and their mechanism of action, we shortly focus on our own contributions to understand the structural equilibrium, high affinity and selectivity of the interaction between this specific RNA sequence and the largest and most complex cellular metabolite, coenzyme B-12. or natural metabolites like coenzyme B 12 are in the center of interest, but also other metal ions, complexes thereof and B 12 derivatives are applied with the aim to understand the largely unknown and manifold non-covalent interactions with nucleic acids. We apply a multitude of techniques, including potentiometric pH titrations, NMR spectroscopy, X-ray crystallography, gel electrophoresis and single molecule FRET experiments. Here we briefly summarize each of our research topics emphasizing the interaction of coenzyme B 12 and its derivatives with the btuB riboswitch of E. coli. This highly conserved sequence, found in the 5'-untranslated region (5'-UTR) of the btuB mRNA, is involved in the regulation of the btuB protein expression. After a summary on the historical discovery of such riboswitches and their mechanism of action, we shortly focus on our own contributions to understand the structural equilibrium, high affinity and selectivity of the interaction between this specific RNA sequence and the largest and most complex cellular metabolite, coenzyme B 12 .

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Divalent metal ions promote the formation of the 5′-splice site recognition complex in a self-splicing group II intron

Cited by 20 publications

References 60 publications

Cation-induced kinetic heterogeneity of the intron–exon recognition in single group II introns

Cation-induced kinetic heterogeneity of the intron–exon recognition in single group II introns

The Role of Mg(II) in DNA Cleavage Site Recognition in Group II Intron Ribozymes

Shaping RNA Structures with Metal Ions and Metal Ion Complexes

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