Fig. 3.
Formation of a stable closed-loop structure on a capped and polyadenylated mRNA in the presence of an 80S complex requires Pab1p interactions with eIF4G, mRNA, and Sup35p. (a) Toeprinting analyses of miniUAA1 mRNA in Pab1p-defective, wild-type, or pbp1Δ extracts. (b) Sensitivity to cap analog in an eIF4G1 mutant incapable of Pab1p interaction. (c) sup35-R419G and sup45-2 mutants show sensitivity to cap analog and additional toeprint bands upstream of the initiator AUG. (d) Sensitivity to cap analog is independent of the termination event. (e, f) Cap analog resistance or sensitivity of miniUAA1 mRNA appears at the onset of translation. (g) Sucrose gradient fractionation of miniUAA1 mRNA translated in wild-type and mutant extracts in the absence and presence of cap analog, and in the presence of CHX. In panel g, the values above the horizontal line depict fraction numbers and those below the line denote the respective percentages of mRNA associated with polysomal or non-polysomal fractions.