Radiolabeled RNA Transcription and Purification

Synthesized DNA templates were used in T7 RNA polymerase transcription reactions carried out with the Ambion T7-Megashortscript kit. [α-32P]UTP (800 Ci/mmol; Perkin–Elmer, Waltham, MA, USA) was included in the reaction to label the transcription products [12 (link),25 (link),26 (link),27 (link),28 (link)]. Transcripts of the correct size were purified by electrophoresis on a 10% (wt/vol) denaturing polyacrylamide gel, followed by elution, phenol extraction, and ethanol precipitation. The same conditions were used for all of the reactions: 25 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 100 mM NaCl, and 10% (vol/vol) glycerol, 20 fmol of substrate, and equimolar amounts of tRNA endonuclease (2 μM). The reactions were incubated at 30 °C or 65 °C for 8 min. Aliquots were pooled at 2 min intervals, the reaction was stopped by phenol extraction and the ethanol precipitated [12 (link)]. The products were separated on 10% (wt/vol) denaturing polyacrylamide gels and analyzed on a Molecular Dynamics model Storm 860 PhosphorImager using ImageQuant software, version 4. Local average background was corrected, and the fraction cleaved was calculated by the ratio of cleaved product to the sum of the cleaved product plus uncleaved substrate [25 (link)].