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Methods:Tritium Release Assay

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Purpose

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This is an assay to detect tRNA pseudouridine synthase activity based on the release of 3H from an tRNA substrate that contains [5-3H]-Ura.[1] By changing the RNA substrate, the assay can be used to detect other enzymes that modify the 5 position of uracil in RNA, such as rRNA pseudouridine synthases [2] and (Uracil-5-)methyltransferases.[3]

Pseudouridine in RNA is synthesized from uridine via the action of Ψ synthases.

Materials Needed

Protocol

"The tritium release assay was a modification of a reported procedure.[3] A typical 100 μL reaction contained 2 nM ΨSI and 1.5 μM [5-3H]Ura-tRNA (2.0 × 104 dpm/pmol) in TNE buffer (20 mM Tris·Cl, pH 8.0, 0.1 M NH4Cl, and 2 mM DTT). Reactions were incubated at 15 or 22 °C. Aliquots of 18 μL were removed at time intervals up to 20 min and quenched with 1 mL of 5% Norit A in 0.1 N HCl. The mixtures were centrifuged for 5 min, and the supernatants were again treated with 0.5 mL of 5% Norit A in 0.1 N HCl. Mixtures were centrifuged for 10 min, the supernatants were filtered through glass wool, and 1.0 mL of each filtrate was counted in 5 mL of Aquasol-2 (New England Nuclear)."[1]

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References

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  1. 1.0 1.1 Huang, L et al. (1998) A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst. Biochemistry 37 344-51 PubMed EcoliWiki page
  2. Huang, L et al. (1998) Identification of two Escherichia coli pseudouridine synthases that show multisite specificity for 23S RNA. Biochemistry 37 15951-7 PubMed EcoliWiki page
  3. 3.0 3.1 Santi, DV & Hardy, LW (1987) Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis. Biochemistry 26 8599-606 PubMed EcoliWiki page