/pdf/functional-nucleic-acid-sensors-k8tlieshyr.pdf

Functional Nucleic Acid Sensors

An in vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson–Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (kcat/Km) of ≈109 M−1⋅min−1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg2+ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1 gag/pol, env, vpr, tat, and nef mRNAs.

https://www.pnas.org/content/pnas/94/9/4262.full.pdf

A General Purpose RNA-Cleaving DNA Enzyme

We present the first complete high-resolution map of nucleosome occupancy across the whole Saccharomyces cerevisiae genome, identifying over 70,000 positioned nucleosomes occupying 81% of the genome. On a genome-wide scale, the persistent nucleosome-depleted region identified previously in a subset of genes demarcates the transcription start site. Both nucleosome occupancy signatures and overall occupancy correlate with transcript abundance and transcription rate. In addition, functionally related genes can be clustered on the basis of the nucleosome occupancy patterns observed at their promoters. A quantitative model of nucleosome occupancy indicates that DNA structural features may account for much of the global nucleosome occupancy.

A high-resolution atlas of nucleosome occupancy in yeast.

https://www.cell.com/cell/pdf/S0092-8674(12)01546-2.pdf

A Decade of Riboswitches

The identification of nuclease-hypersensitive sites in an active globin gene1 and in the 5′ regions of fruit fly heat shock genes2 first suggested that chromatin changes accompany gene regulation in vivo. Here we present evidence that the basic repeating units of eukaryotic chromatin, nucleosomes, are depleted from active regulatory elements throughout the Saccharomyces cerevisiae genome in vivo. We found that during rapid mitotic growth, the level of nucleosome occupancy is inversely proportional to the transcriptional initiation rate at the promoter. We also observed a partial loss of histone H3 and H4 tetramers from the coding regions of the most heavily transcribed genes. Alterations in the global transcriptional program caused by heat shock or a change in carbon source resulted in an increased nucleosome occupancy at repressed promoters, and a decreased nucleosome occupancy at promoters that became active. Nuclease-hypersensitive sites occur in species from yeast to humans and result from chromatin perturbation3,4,5. Given the conservation of sequence and function among components of both chromatin and the transcriptional machinery, nucleosome depletion at promoters may be a fundamental feature of eukaryotic transcriptional regulation.

Evidence for nucleosome depletion at active regulatory regions genome-wide.

The natural RNA enzymes catalyse phosphate-group transfer and peptide-bond formation. Initially, metal ions were proposed to supply the chemical versatility that nucleotides lack. In the ensuing decades, structural and mechanistic studies have substantially altered this initial viewpoint. Whereas self-splicing ribozymes clearly rely on essential metal-ion cofactors, self-cleaving ribozymes seem to use nucleotide bases for their catalytic chemistry. Despite the overall differences in chemical features, both RNA and protein enzymes use similar catalytic strategies.

The catalytic diversity of RNAs.

Statistical positioning of nucleosomes by specific protein-binding to an upstream activating sequence in yeast.

Structure and function of the hairpin ribozyme

The "hammerhead" RNA self-cleaving domain can be assembled from two RNA molecules: a large (approximately 34 nucleotide) ribozyme RNA containing most of the catalytically essential nucleotides and a small (approximately 13 nucleotide) substrate RNA containing the cleavage site Four such hammerheads that contained identical catalytic core sequences but differed in the base composition of the helices that are involved in substrate binding had been reported to vary in cleavage rates by more than 70-fold under similar reaction conditions Steady-state kinetic analyses reveal that kcat values are nearly the same for these hammerheads but Km values vary nearly 60-fold The substrates for reactions having high Km values form aggregates that are virtually nonreactive These observations demonstrate that the secondary structure of substrate RNA can be a major determinant of hammerhead catalytic efficiency

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Substrate sequence effects on "hammerhead" RNA catalytic efficiency.

The hammerhead catalytic RNA effects cleavage of the phosphodiester backbone of RNA through a transesterification mechanism that generates products with 2'-3'-cyclic phosphate and 5'-hydroxyl termini A minimal kinetic mechanism for the intermolecular hammerhead cleavage reaction includes substrate binding, cleavage, and product release Elemental rate constants for these steps were measured with six hammerhead sequences Changes in substrate length and sequence had little effect on the rate of the cleavage step, but dramatic differences were observed in the substrate dissociation and product release steps that require helix-coil transitions Rates of substrate binding and product dissociation correlated well with predictions based on the behavior of simple RNA duplexes, but substrate dissociation rates were significantly faster than expected Ribozyme and substrate alterations that eliminated catalytic activity increased the stability of the hammerhead complex These results suggest that substrate destabilization may play a role in hammerhead catalysis

Martha J. Fedor

Papers

The catalytic diversity of RNAs.

Statistical positioning of nucleosomes by specific protein-binding to an upstream activating sequence in yeast.

Structure and function of the hairpin ribozyme

Substrate sequence effects on "hammerhead" RNA catalytic efficiency.

Kinetics of intermolecular cleavage by hammerhead ribozymes