Figure 3.

Cap–poly(A) synergy on a cellular mRNA as a function of the degree of extract depletion. Aliquots (A–E) of ribosome-depleted RRL were prepared under different centrifugation conditions as follows using 1.1 ml volumes of RRL: centrifugation in a fixed-angle TL100 rotor at 90 000 r.p.m. for 25 min (extract A), or 90 000 r.p.m. for 15 min (extract F). Aliquots B through E were derived by mixing aliquots A and F, in the ratios 98.5:1.5, 97:3, 95.5:4.5 and 93:7, respectively. Translation reactions containing ribosome-depleted RRL extracts A–E were programmed with 6.3 µg/ml of pOp24-derived mRNAs transcribed in the form indicated above each lane, and contained 2.5% by volume of HeLa cell S10 extract. Final concentrations of added KCl and MgCl₂ were 130 and 0.93 mM, respectively. Translation reactions were analysed as described in Materials and Methods; the autoradiograph of the dried 23% polyacrylamide gel is shown. Translation efficiency was determined densitometrically as described in Materials and Methods, and is plotted in arbitrary units. Closed and open squares represent capped, polyadenylated and capped, non-polyadenylated mRNAs, respectively. Stimulation by poly(A) is also plotted (open circles).