Exploration of the Transition State for Tertiary Structure Formation between an RNA Helix and a Large Structured RNA

Bartley, Laura E.; Zhuang, Xiaowei; Das, Rhiju; Chu, Steven; Herschlag, Daniel

doi:10.1016/s0022-2836(03)00272-9

Cited by 105 publications

(186 citation statements)

References 115 publications

(140 reference statements)

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“…The previous results and the analysis of the +1U effect suggest that the interactions of the U (+1) containing substrate with the L-21 ScaI are fortuitous, providing another example of RNA's conformational promiscuity (e.g., (30,55,77,78). The promiscuous interaction can be overcome by occupying other portions of the active site in their cognate interactions with G and by immersing the U•U pair within a duplex, effects that have implications for understanding the origins of tertiary structure specificity in RNA (58).…”

Section: Probing the Effect Of A U•u Mismatch After The Cleavage Sitementioning

confidence: 86%

“…Single-molecule FRET measurements at 22 °C have shown that K tert for the L-21 ScaI and the L-16 ScaI (with all base pairs in the P1 extension formed) are similar with values of 22 ± 5 and 32 ± 4, respectively (55). These data are consistent with bulk measurements using modified substrate analogs at 30 °C (K tert = 15 and 23 for binding to the L-21 ScaI and the L-16 ScaI ribozyme, respectively, data not shown).…”

Section: No Significant Contribution To 5 ′ -Splice Site Analog (S) Bmentioning

confidence: 88%

“…Modification of the 2′-hydroxyl group at position -3 to a 2′-methoxy group destabilizes the docked ribozyme/substrate duplex conformation while not affecting the stability of the P1 duplex (7,35,55). Thus, substrate analogs with this modification approximate the affinity of the duplex between substrate and IGS (for K tert < 1, ,Eq.2B).…”

Section: Measurement Of the Equilibrium Constant For Dockingmentioning

confidence: 99%

See 2 more Smart Citations

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

2007

Self Cite

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Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogs, catalyzes a reaction analogous to the first step of self-splicing, in which a 5′-splice site analog (S) and guanosine (G) are converted into a 5′-exon analog (P) and GA. This ribozyme preserves the active site but lacks a short 5′-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5′-splice site in the first step of self-splicing and P10 forms at the 3′-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5′-and 3′-splice site analogs that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogs that do not form additional base pairs with the IGS extension. Analysis of a U•U pair immediately 3′ to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5′-splice site analogs that form only a subset of base pairs reveal that inclusion of the conserved G•U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G•U step. These results suggest a previously unrecognized role of the G•U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.The group I intron from Tetrahymena thermophila, the first catalytic RNA to be discovered, folds into a specific structure and performs a self-splicing reaction that includes chemical steps as well as RNA conformational rearrangements. This reaction is simple, relative to pre-mRNA † This work was supported by NIH grant GM49243. K.K. was supported in part by a predoctoral fellowship from the Boehringer Ingelheim NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript splicing and other RNA-mediated processes, and may serve as a tractable system to learn more about RNA catalysis and its functional conformational change...

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Section: Probing the Effect Of A U•u Mismatch After The Cleavage Sitementioning

confidence: 86%

Section: No Significant Contribution To 5 ′ -Splice Site Analog (S) Bmentioning

confidence: 88%

Section: Measurement Of the Equilibrium Constant For Dockingmentioning

confidence: 99%

See 1 more Smart Citation

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

2007

Self Cite

View full text Add to dashboard Cite

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“…A scanning confocal fluorescence microscope (SCFM) that is sensitive enough to detect single fluorescent molecules can be used to observe single-pair fluorescence resonance energy transfer (spFRET) [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. Fluorescence resonance energy transfer (FRET) is a spectroscopic process in which non-radiative energy transfer occurs between an excited dipole (the donor) and another dipole (the acceptor) that has an absorption spectrum that overlaps the emission spectrum of the donor [46].…”

Section: Introductionmentioning

confidence: 99%

“…Changes in the conformation of the molecule are detected as changes in the energy transfer between the donor and acceptor molecule. In this fashion, single molecular-pair FRET has been used to study, e.g., nanometer conformational motions in individual RNA molecules [29][30][31][32][33][34][35], individual DNA molecules [36][37][38][39][40][41] and individual helicases and other motor proteins on DNA [42][43][44][45]. By following changes in FRET efficiency over time, it is possible to observe nanometer distance changes due to macromolecular rearrangements.…”

Section: Introductionmentioning

confidence: 99%

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

Zheng¹,

Goldner²,

Leuba³

2007

Methods

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Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/ acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

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Cross‐Reactive Sensor Arrays for the Detection of Peptides in Aqueous Solution by Fluorescence Spectroscopy

Rochat

Gao

Qian

et al. 2009

Chemistry A European J

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A simple but powerful method for the sensing of peptides in aqueous solution has been developed. The transition‐metal complexes [PdCl2(en)], [{RhCl2Cp*}2], and [{RuCl2(p‐cymene)}2] were combined with six different fluorescent dyes to build a cross‐reactive sensor array. The fluorescence response of the individual sensor units was based on competitive complexation reactions between the peptide analytes and the fluorescent dyes. The collective response of the sensor array in a time‐resolved fashion was used as an input for multivariate analyses. A sensor array comprised of only six metal–dye combinations was able to differentiate ten different dipeptides in buffered aqueous solution at a concentration of 50 μM. Furthermore, the cross‐reactive sensor could be used to obtain information about the identity and the quantity of the pharmacologically interesting dipeptides carnosine and homocarnosine in a complex biological matrix, such as deproteinized human blood serum. The sensor array was also able to sense longer peptides, which was demonstrated by differentiating mixtures of the nonapeptide bradykinin and the decapeptide kallidin.

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Exploration of the Transition State for Tertiary Structure Formation between an RNA Helix and a Large Structured RNA

Cited by 105 publications

References 115 publications

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

Cross‐Reactive Sensor Arrays for the Detection of Peptides in Aqueous Solution by Fluorescence Spectroscopy

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