The luciferase constructs used in translation assays consisting of the firefly luciferase gene (luc2, Promega) flanked by the indicated viral genomic 5′ and 3′ UTRs [32 (link)] were linearized with Sma I and transcribed using the MEGAscript kit (Ambion). CAluc, which is a capped transcript containing nonviral UTRs described previously [73 (link)], was linearized with EcoICRI, transcribed with the MEGAscript kit (Ambion) and post-transcriptionally capped using the T7 mScript Standard mRNA Production System (Cell Script).
For trans-inhibition and structure probing experiments, the PTE sequences from the indicated viruses present in the universal SHAPE cassette [15 (link),32 (link)] were linearized with either HpaI (giving only PTE transcript for trans-inhibition studies) or SmaI (which adds a 3′-terminal extension on the PTE providing a primer binding site for SHAPE experiments) and transcribed in vitro using the MEGAshortscript kit (Ambion). All transcripts were purified by phenol/chloroform extraction and ethanol precipitation. RNA concentrations were determined spectrophotometrically and integrity was verified by 0.8% agarose gel electrophoresis.
For trans-inhibition and structure probing experiments, the PTE sequences from the indicated viruses present in the universal SHAPE cassette [15 (link),32 (link)] were linearized with either HpaI (giving only PTE transcript for trans-inhibition studies) or SmaI (which adds a 3′-terminal extension on the PTE providing a primer binding site for SHAPE experiments) and transcribed in vitro using the MEGAshortscript kit (Ambion). All transcripts were purified by phenol/chloroform extraction and ethanol precipitation. RNA concentrations were determined spectrophotometrically and integrity was verified by 0.8% agarose gel electrophoresis.
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