Escherichia coli Ribonuclease III: Affinity Purification of Hexahistidine-Tagged Enzyme and Assays for Substrate Binding and Cleavage

Amarasinghe, Asoka K.; Calin‐Jageman, Irina; Harmouch, Ahmed; Sun, Weimei; Nicholson, Allen W.

doi:10.1016/s0076-6879(01)42542-0

Cited by 60 publications

(151 citation statements)

References 43 publications

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“…In contrast, Escherichia coli RNase III (EC3.1.24) can digest dsRNA very efficiently into short pieces with the same end structures as siRNA, 5Ј phosphate͞3Ј hydroxyl termini and 2-to 3-nt 3Ј overhangs (17). These end structures of siRNA are reported to be important for RNAi activity (18).…”

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confidence: 99%

“…These end structures of siRNA are reported to be important for RNAi activity (18). In addition, large amounts of soluble recombinant E. coli RNase III protein can be obtained (17). These attributes make E. coli RNase III a promising enzyme for preparing siRNAs in vitro.…”

mentioning

confidence: 99%

“…Exhaustive cleavage of dsRNA by E. coli RNase III leads to duplex products averaging 12-15 bp in length (17). These short dsRNA are unable to trigger an RNAi response in mammalian cells (ref.…”

mentioning

confidence: 99%

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Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells

Yang

Buchholz

Huang

et al. 2002

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

282

182

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Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of Escherichia coli RNase III to cleave doublestranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in Drosophila S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells. esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.

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confidence: 99%

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confidence: 99%

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Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells

Yang

Buchholz

Huang

et al. 2002

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

282

182

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“…Sequencing of cloned fragment was performed using the ABI 3700 sequencer (Applied Biosystems). The cloned gene was properly manipulated for further purification and enzymatic assay purposes as described [45].…”

Section: Pcr Amplificationsmentioning

confidence: 99%

“…The E. coli RNAse III gene was properly cloned within Nco1 and BamH1 of pIVEX2.4a (Roche Applied Science, Indianapolis, IN 46250 United States) to produce and purified the recombinant protein as described [45] in the form of 6x(His)-RNAse III. Double stranded RNase activity of recombinant protein form was performed basically with the same method we used for S. pombe strain 428-4-1 but with minor variations [47].…”

Section: Enzymatic Assay Of Recombinant E Coli Rnase IIImentioning

confidence: 99%

Non-linear models based on simple topological indices to identify RNase III protein members

Agüero-Chapin

Riva

Ruiz

et al. 2011

Journal of Theoretical Biology

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Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB

et al. 2015

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Ribonuclease III is a conserved bacterial endonuclease that cleaves double-stranded(ds) structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control that in turn confer global post-transcriptional regulation. The Escherichia coli macrodomain protein YmdB directly interacts with RNase III, and an increase in YmdB amount in vivo correlates with a reduction in RNase III activity. Here, a computational-based structural analysis was performed to identify atomic-level features of the YmdB-RNase III interaction. The docking of monomeric E. coli YmdB with a homology model of the E. coli RNase III homodimer yields a complex that exhibits an interaction of the conserved YmdB residue R40 with specific RNase III residues at the subunit interface. Surface Plasmon Resonance (SPR) analysis provided a KD of 61 nM for the complex, corresponding to a binding free energy (ΔG) of −9.9 kcal/mol. YmdB R40 and RNase III D128 were identified by in silico alanine mutagenesis as thermodynamically important interacting partners. Consistent with the prediction, the YmdB R40A mutation causes a 16-fold increase in KD (ΔΔG = +1.8 kcal/mol), as measured by SPR, and the D128A mutation in both RNase III subunits (D128A/D128'A) causes an 83-fold increase in KD (ΔΔG = +2.7 kcal/mol). The greater effect of the D128A/D128'A mutation may reflect an altered RNase III secondary structure, as revealed by CD spectroscopy, which also may explain the significant reduction in catalytic activity in vitro. The features of the modeled complex relevant to potential RNase III regulatory mechanisms are discussed.

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Escherichia coli Ribonuclease III: Affinity Purification of Hexahistidine-Tagged Enzyme and Assays for Substrate Binding and Cleavage

Cited by 60 publications

References 43 publications

Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells

Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells

Non-linear models based on simple topological indices to identify RNase III protein members

Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB

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