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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: Virology. 2009 Jan 7;385(1):245–260. doi: 10.1016/j.virol.2008.11.041

Fig. 7.

Fig. 7

Binding of eEF1A to the replication silencer sequence of the TBSV (+)repRNA in vitro. (A) Gel mobility shift assay was performed with the 32P-labeled WT-SL1/SL2/SL3(+) RNA probe. The SL1/SL2/SL3 sequence represents the 86 nt 3′ terminal, highly structured sequence of the TBSV (+)RNA with two known cis-acting elements: the replication silencer element required for the assembly of the viral replicase and gPR that is an essential promoter for initiation of minus-strand synthesis and it is also required for replicase assembly. The mutated/deleted bases are indicated with black boxes. The recombinant eEF1A purified from yeast as GST-fusion protein was tested in three different dilutions: 2.0, 0.8 and 0.4 μg. (B) Template competition assay to test the binding of eEF1A to the viral template. Gel mobility shift assay was performed with the 32P-labeled WT-SL1/SL2/SL3(+) RNA probe and purified eEF1A (as in panel A), whereas the unlabeled competitor RNA was used in 5x, 20x and 100x excess over the labeled one as shown. Template gPR-CCC-UUU(+) has the 3′ terminal three Cs changed to three Us (not shown).