Figure 3. eEF1A promotes the initiation by the TCV RdRp during minus-strand synthesis.

(A) Purified eEF1A was added to the TCV RdRp assay as shown. The TBSV (+)RNA template was the short 3′ end region (SL1/SL2/SL3), which contain the promoter region (SL1) for initiation and the replication silencer element (within SL3) that down-regulates initiation. The second template was SL1m with a point mutation within the promoter sequence, which is being used more efficiently by the TCV RdRp in vitro. Note that eEF1A has been shown to bind to the replication silencer element. The RdRp assay had two steps: first, the shown components were incubated at room temperature to facilitate their interaction, followed 5 min latter the addition of the shown component and the ribonucleotides to start RNA synthesis. The RdRp activity in samples containing the template RNA and the RdRp were chosen as 100% (lanes 3–4). (B) Detection of abortive RdRp products in the in vitro assay. 15% PAGE/UREA gel was used to resolve the 4–10 nt long products produced during initiation followed by rapid termination. Note that abortive RNA products are characteristic products for RNA polymerases that initiate de novo (in the absence of a traditional primer). (C) Lack of stimulation of 3′-terminal extension by eEF1A in vitro. The template RNAs (shown schematically) contain a common artificial hairpin structure at the 3′ end that facilitates 3′-TEX by the TCV RdRp. The black bar represents 3 different sequences in the three constructs, derived from RIV(+)(includes SL1/SL2/SL3 sequences), RIII(−) and RIII(+) of DI-72 RNA, respectively. The gel image shows the results of 3′-TEX in the presence of 0 or 1 µg eEF1A as shown in a TCV RdRp assay.