Biotin-labeled JFH1 nt 1 360 together with rhRBM24 or BSA were incubated in RRL at 30°C for 15 min. The ribosome complexes were separated by sucrose gradient ultracentrifugation.
(C) The RNA fragment containing HCV IRES and the firefly luciferase coding sequence was incubated with either rhRBM24 or BSA in RRL at 30°C for 15 min. Luciferase activity was determined with Steady-Glo®.
This significantly shortened GLV IRES in RRL is only 210 nts and is approximately twice as active as the full length GLV IRES in RRL (Fig. 3, G).
Our study shows that GLV IRES is functional in RRL system despite the evolutionary divergence between Giardia and rabbit.
In vitro translations of FF-HCV-Ren in RRL (Promega) were performed according to the manufacturer's instructions.
However, HIV-1 PR was not able to hamper translation of luc mRNA in RRL (Figure 3C).
By contrast, poly(A) tail did not contribute substantially to initiation of translation in RRL (Figure 3C).
The Fluc activity was completely lost, suggesting a real, albeit limited, function of this truncated GLV IRES in RRL.
These structures were originally postulated to accommodate the recruited 40S ribosomal subunit in Giardia [44] and may still serve the same function in RRL.
Control reactions showed that RNase A inhibited these reactions, indicating that DNA synthesis was catalyzed by telomerase and not by contaminating polymerases present in RRL.
Interestingly, the results also indicated that the Dbox is apparently involved in the IRES activity in RRL (Fig. 3, compare E and F).
