FIG. 5.

(A) Toeprinting of 48S complexes assembled on mRNAs Td3, bG, and Td6 with addition of different amounts of native eIF4B. The amounts of eIF4B per standard volume of the reconstitution mixture (20 μl) were as follows: lanes 2 and 7, 11 to 0.025 μg, lanes 3 and 8, 12 to 0.05 μg, lanes 4 and 9, 13 to 0.1 μg, lanes 5 and 10, 14 to 0.2 μg, and lane 6, 0.3 μg. The yields of 48S complexes for Td6 and bG with 0.3 μg of eIF4B were the same as with 0.2 μg of eIF4B, and the respective lanes are not presented in the figure. No exogenous eIF4B was added to the samples corresponding to lanes 2, 7, and 11. The indicated amount of eIF4B in these samples is accounted for by contamination of affinity purified eIF4F with eIF4B. It was estimated by quantitative Western blotting as 0.05 μg of eIF4B/1 μg of eIF4F. Lane bG control corresponds to the 48S complex reconstituted with mRNA bG in the absence of Met-tRNAiMet and eIF2. A dideoxynucleotide sequence was generated from pTd3. (B) Dependence of formation of 48S complexes assembled on mRNAs bG, Td3, and Td6 on the molar concentration of eIF4B in the reconstitution mixture. The yield of 48S complexes (radioactivity in toeprint bands) was determined with phosphorimager from data presented in A and presented as the ratio to the maximal yield of the complexes for each mRNA.