Novel spermine–Amino acid conjugates and basic tripeptides enhance cleavage of the hairpin ribozyme at low magnesium ion concentration

Stolze, Karen; Holmes, Stephen C.; Earnshaw, David; Singh, Mandeep; Stetsenko, Dmitry A.; Williams, David M.; Gait, Michael J.

doi:10.1016/s0960-894x(01)00608-4

Cited by 11 publications

(6 citation statements)

References 21 publications

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“…One can measure the PSF by imaging subresolution particles such as fluorescence beads; however, this approach gives limited precision because the sample itself is part of the optical system and thus distorts the PSF (50). To overcome this problem, investigators have developed blind deconvolution (50,51) methods that estimate both the deconvolved image and the PSF from the original images. These methods are computationally intense.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Measurement of Protein Relocalization in Live Cells

Bush

Colman‐Lerner

2013

Biophysical Journal

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Microscope cytometry provides a powerful means to study signaling in live cells. Here we present a quantitative method to measure protein relocalization over time, which reports the absolute fraction of a tagged protein in each compartment. Using this method, we studied an essential step in the early propagation of the pheromone signal in Saccharomyces cerevisiae: recruitment to the membrane of the scaffold Ste5 by activated Gβγ dimers. We found that the dose response of Ste5 recruitment is graded (EC50 = 0.44 ± 0.08 nM, Hill coefficient = 0.8 ± 0.1). Then, we determined the effective dissociation constant (K(de)) between Ste5 and membrane sites during the first few minutes when the negative feedback from the MAPK Fus3 is first activated. K(de) changed during the first minutes from a high affinity of < 0.65 nM to a steady-state value of 17 ± 9 nM. During the same period, the total number of binding sites decreased slightly, from 1940 ± 150 to 1400 ± 200. This work shows how careful quantification of a protein relocalization dynamic can give insight into the regulation mechanisms of a biological system.

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Section: Discussionmentioning

confidence: 99%

Quantitative Measurement of Protein Relocalization in Live Cells

Bush

Colman‐Lerner

2013

Biophysical Journal

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“…As shown by Gait and co‐workers (25, 26) and by our laboratory (21), efficient hairpin ribozyme cleavage can be affected by millimolar concentrations of the tetraamine spermine, even in the absence of metal ions. This observation later led to the synthesis of hairpin ribozyme–spermine conjugates with the polyamine covalently attached to the C5 position of specific uridines in loop B and helix 4 (27).…”

Section: Structural Variants Of the Hairpin Ribozymementioning

confidence: 62%

The many faces of the hairpin ribozyme: Structural and functional variants of a small catalytic rna

et al. 2011

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SummaryThe hairpin ribozyme is a small catalytic RNA that has been reengineered resulting in a number of variants with extended or even new functions. Thus, manipulation of the hairpin ribozyme structure has allowed for activity control by external effectors, namely oligonucleotides, flavine mononucleotide, and adenine. Hairpin ribozyme-derived twin ribozymes that mediate RNA fragment exchange reactions as well as self-processing hairpin ribozymes were designed. Furthermore, several hairpin ribozyme variants have been engineered for knock down of specific RNA substrates by adapting the substrate-binding domain to the specific target sequence. This review will focus on hairpin ribozymes possessing structural extensions/variations and thus functionally differing from the parent hairpin ribozyme.

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“…In another study, it was shown that spermine in combination with near physiological Mg 21 concentrations can actually enhance the activity of certain engineered motifs (154). Similarly to previously described strategies, certain spermine-amino acid conjugates stimulated hairpin cleavage at low magnesium ion concentration (155).…”

Section: Polyaminesmentioning

confidence: 72%

Insights into functional modulation of catalytic RNA activity

et al. 2008

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SummaryRNA molecules play critical roles in cell biology, and novel findings continuously broaden their functional repertoires. Apart from their well-documented participation in protein synthesis, it is now apparent that several noncoding RNAs (i.e., micro-RNAs and riboswitches) also participate in the regulation of gene expression. The discovery of catalytic RNAs had profound implications on our views concerning the evolution of life on our planet at a molecular level. A characteristic attribute of RNA, probably traced back to its ancestral origin, is the ability to interact with and be modulated by several ions and molecules of different sizes. The inhibition of ribosome activity by antibiotics has been extensively used as a therapeutical approach, while activation and substrate-specificity alteration have the potential to enhance the versatility of ribozyme-based tools in translational research. In this review, we will describe some representative examples of such modulators to illustrate the potential of catalytic RNAs as tools and targets in research and clinical approaches.

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Novel spermine–Amino acid conjugates and basic tripeptides enhance cleavage of the hairpin ribozyme at low magnesium ion concentration

Cited by 11 publications

References 21 publications

Quantitative Measurement of Protein Relocalization in Live Cells

Quantitative Measurement of Protein Relocalization in Live Cells

The many faces of the hairpin ribozyme: Structural and functional variants of a small catalytic rna

Insights into functional modulation of catalytic RNA activity

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