RNA-Protein Cross-Linking and Primer Extension

A mixture (8 μl) containing RnaG120 (0.5 pmol) and the [³²P]-labeled G+1H oligonucleotide (2 pmol) was heated at 90°C for 1 min to denature RNA. Annealing of the primer with RnaG120 was carried out at 32°C for 5 min in buffer A (see above) in the presence or absence of VirF. The mixture was transferred to an ice-cold plate and U.V. irradiated for 1 min using the GS Gene-linker BioRad (180 mJ, 254 nm bulbs at ∼12 cm from the U.V. source). Finally the cross-linked RNA was primer-extended using the AMV Reverse Transcriptase as previously described (Giangrossi et al., 2010 (link)).