FIG 7.
Translocation of the IAPV IRES from the ribosomal A to P sites in the presence of HygB and LTM. Bicistronic RNAs containing the wild-type IGR IRES were incubated in the presence of the HygB (1 μM) and LTM (5 μM) in RRL for 15 min (lane 1). (Lanes 2–9) LTM was added at the indicated times after starting the reaction in the presence of HygB. Primer extension analysis was carried out in the presence of [32P]-dATP. Primer extension products were resolved by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Quantitations of toeprints +17 and +14 are shown below at the indicated times of addition of LTM. Fraction of toeprints was calculated by the radioactive counts for toeprint +17 or +14 divided by the total counts of toeprints +14 and +17 which represent the binding and translocation of ribosomes on the IAPV IRES. Shown is a representative gel and quantitation from three independent experiments.