In-gel fluorescence probing of RNA–RNA interactions

Dayie, T. Kwaku; Gumbs, Orlando H.; Eldho, Nadukkudy V.; Seetharaman, Mahadevan; Thompson, Michalynn

doi:10.1016/j.ab.2006.12.020

Cited by 3 publications

(8 citation statements)

References 15 publications

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“…To test this hypothesis, we used a splicing reaction buffer of 40 mM MOPS (X + ) (pH 7.50) and 10 mM MgCl 2 containing 100 mM XCl, where X = Li + , Na + , K + , and Cs + . To visualize the cleaved fluorescently labeled substrate (Alexa647-E1E2), we used a previously developed fluorescence in-gel method , . To ensure there are no competing monovalent ions, we dialyzed D1235 extensively against water for 24 h and then against MOPS (X + ) (pH 7.50) for 12 h.…”

Section: Methodsmentioning

confidence: 99%

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Probing Adenine Rings and Backbone Linkages Using Base Specific Isotope-Edited Raman Spectroscopy: Application to Group II Intron Ribozyme Domain V

et al. 2010

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Raman difference spectroscopy is used to probe the properties of a 36-nt RNA molecule, “D5”, which lies at the heart of the catalytic apparatus in group II introns. For D5 that has all its adenine residues labeled with 13C and 15N, and utilizing Raman difference spectroscopy, we identify the conformational sensitive -C-O-P-O-C- stretching modes of the unlabeled bonds adjacent to adenine bases, as well as the adenine ring modes themselves. The phosphodiester modes can be assigned to individual adenine residues based on earlier NMR data. The effect of Mg2+ binding was explored by analyzing the Raman difference spectra for [D5 + Mg2+] minus [D5 no Mg2+], for D5 unlabeled, or D5 labeled with 13C/15N-enriched adenine. In both sets of data we assign differential features to G ring modes perturbed by Mg2+ binding at the N7 position. In the A labeled spectra we attribute a Raman differential near 1450 cm−1 and changes of intensity at 1296 cm−1 to Mg binding at the N7 position of adenine bases. The A and G bases involved in Mg2+ binding again can be identified using earlier NMR results. For the unlabeled D5, a change in the C-O-P-O-C stretch profile at 811 cm−1 upon magnesium binding is due to a “tightening up” (in the sense of a more rigid molecule with less dynamic interchange among competing ribose conformers) of the D5 structure. For adenine labeled D5, small changes in the adenine backbone bond signatures in the 810 – 830 cm−1 region suggest small conformational changes occur in the tetraloop and bulge regions upon binding of Mg2+. The PO2− stretching vibration, near 1100 cm−1, from the non-bridging phosphate groups, probes the effect of Mg2+-hydrate inner-sphere interactions that cause an up-shift. In turn, the up-shift is modulated by the presence of monovalent cations since in the presence of Na+ and Li+ the up-shift is (23±2 cm−1) while in the presence of K+ and Cs+ it is (13±3 cm−1), a finding that correlates with the differences in hydration radii. These subtle differences in electrostatic interactions may be related to observed variations in catalytic activity. For a reconstructed ribozyme comprising domains 1–3 (D123) connected in cis plus domain 5 (D5) supplied in trans, cleavage of spliced exon substrates in the presence of magnesium and K+ or Cs+ is more efficient than that in the presence of magnesium with Na+ or Li+.

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Section: Methodsmentioning

confidence: 99%

“…At each time point (0, 30, 60, 120, 180 min), a 5 μL aliquot of the reaction is quenched using the stopping buffer (100 mM EDTA, pH 8.0, 10% glycerol). The extent of the cleavage was visualized on a Molecular Imager (Storm imager; Molecular Dynamics), and the uncleaved and cleaved substrate intensities were quantified using Image Quant , .…”

Section: Methodsmentioning

confidence: 99%

Probing Adenine Rings and Backbone Linkages Using Base Specific Isotope-Edited Raman Spectroscopy: Application to Group II Intron Ribozyme Domain V

et al. 2010

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“…Determining the 3D architecture of the group II intron is the beginning process to answer this question, and in turn this requires defining an experimentally well-behaved model system. We recently reconstructed a group II intron from Pylaiella littoralis into a highly active form by combining an unstructured 22 nt substrate, a 36 nt domain 5 (D5) hairpin, and a 493 nt domains 1 through 3 (D123) [ 109 – 112 ]. This new tripartite ribozyme system functions under low salt conditions attractive for NMR structural studies and has enabled us to determine a very high resolution NMR structure of the catalytic lynchpin, D5 [ 110 ].…”

Section: Tackling Sizeable Problems In Rna Biophysical Chemistrymentioning

confidence: 99%

“…This new tripartite ribozyme system functions under low salt conditions attractive for NMR structural studies and has enabled us to determine a very high resolution NMR structure of the catalytic lynchpin, D5 [ 110 ]. We have also quantitatively assayed the binding of each component using a fluorescence native gel mobility shift assay we developed in conjunction with fluorescent anisotropy measurements [ 109 , 112 ]. Parenthetically, this fluorescent method takes advantage of a multicolor fluorescent-based gel assay to directly monitor RNA-RNA interactions, without the need to use hazardous 32 P labeled probes [ 109 , 112 – 113 ].…”

Section: Tackling Sizeable Problems In Rna Biophysical Chemistrymentioning

confidence: 99%

“…We have also quantitatively assayed the binding of each component using a fluorescence native gel mobility shift assay we developed in conjunction with fluorescent anisotropy measurements [ 109 , 112 ]. Parenthetically, this fluorescent method takes advantage of a multicolor fluorescent-based gel assay to directly monitor RNA-RNA interactions, without the need to use hazardous 32 P labeled probes [ 109 , 112 – 113 ]. Using pre-steady state-kinetic experiments, we have shown that the cleavage reaction is pH-dependent, suggesting that the chemical step is rate limiting [ 109 ].…”

Section: Tackling Sizeable Problems In Rna Biophysical Chemistrymentioning

confidence: 99%

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Key Labeling Technologies to Tackle Sizeable Problems in RNA Structural Biology

Dayie

2008

IJMS

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Abstract:The ability to adopt complex three-dimensional (3D) structures that can rapidly interconvert between multiple functional states (folding and dynamics) is vital for the proper functioning of RNAs. Consequently, RNA structure and dynamics necessarily determine their biological function. In the post-genomic era, it is clear that RNAs comprise a larger proportion (>50%) of the transcribed genome compared to proteins (≤ 2%). Yet the determination of the 3D structures of RNAs lags considerably behind those of proteins and to date there are even fewer investigations of dynamics in RNAs compared to proteins. Site specific incorporation of various structural and dynamic probes into nucleic acids would likely transform RNA structural biology. Therefore, various methods for introducing probes for structural, functional, and biotechnological applications are critically assessed here. These probes include stable isotopes such as 2 H, 13 C, 15 N, and 19 F.Incorporation of these probes using improved RNA ligation strategies promises to change the landscape of structural biology of supramacromolecules probed by biophysical tools such as nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and Raman spectroscopy. Finally, some of the structural and dynamic problems that can be addressed using these technological advances are outlined.

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Influence of pH and Mg(ii) on the catalytic core domain 5 of a bacterial group II intron

2017

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RNA molecules fold into complex structures that allow them to perform specific functions. To compensate the relative lack of diversity of functional groups within nucleotides, metal ions work as crucial co-factors. In addition, shifted pKs are observed in RNA, enabling acid-base reactions at ambient pH. The central catalytic domain 5 (D5) hairpin of the Azotobacter vinelandii group II intron undergoes both metal ion binding and pH dependence, presumably playing an important functional role in the ribozyme's reaction. By NMR spectroscopy we have here characterized the metal ion binding sites and affinities for the hairpin's internal G-A mismatch, bulge, and pentaloop. The influence of Mg(ii) and pH on the local conformation of the catalytically crucial region is also explored by fluorescence spectroscopy.

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In-gel fluorescence probing of RNA–RNA interactions

Cited by 3 publications

References 15 publications

Probing Adenine Rings and Backbone Linkages Using Base Specific Isotope-Edited Raman Spectroscopy: Application to Group II Intron Ribozyme Domain V

Probing Adenine Rings and Backbone Linkages Using Base Specific Isotope-Edited Raman Spectroscopy: Application to Group II Intron Ribozyme Domain V

Key Labeling Technologies to Tackle Sizeable Problems in RNA Structural Biology

Influence of pH and Mg(ii) on the catalytic core domain 5 of a bacterial group II intron

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