• 1 M DTT

1 pi

• 30mM potassium phosphate, pH 7.0

5 pi

• 10 mM GTP

5 pi

• 0.25 M Hepes-KOH, pH 7.5

5 pl

• 2 M KCl

1.5 pi

• 60mM magnesium acetate

5 pl

• 10mM ATP

5 pl

• [a-32P] UTP

1 Ml

• mitochondrial extract

10 Ml

• cytosolic RNA substrate (prepared as above)

1 pl

2. Incubate the mixture for 40 min at 27 °C.

3. Stop the reaction by adding 50 pi of 0.5 M sodium phosphate buffer, pH 6.8, 0.5% sodium pyrophosphate, 0.1% SDS.

4. Remove 80 pi aliquots onto DE81 filter discs.

5. Process the discs and determine the retained radioactivity as described in Protocol 9, steps 4-6.

8.4 RNA ligase

An RNA ligase activity was first observed in total cell extracts from T. brucei (71). The relationship of the total cell RNA ligase activity to the mitochondrial activity described in L. tarentolae is not clear. The L. tarentolae RNA ligase activity co-sediments with the kinetoplast-mitochondrion fraction (50). It is solubilized in Triton X-100 and thus can be assayed in mitochondrial extracts. Incubation of cytosolic RNA with mitochondrial TL extract and [<x-32P]UTP under the conditions required for TUTase in the presence of ATP and Mg2+ ions yields a 180nt labelled RNA product whose relative electrophoretic mobility varies with the gel concentration, a behaviour characteristic of circular RNA molecules. RNA ligase is quantitatively assayed by the addition of [a-32P]pCp to the 3' termini of RNA molecules as described in Protocol 13.

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