Fig. 3.

Although not required for initiation, eIF5A directly stimulates protein synthesis in vitro in a hypusine-dependent manner. Translation extracts depleted of UBR5A for 2.5 h were prepared and assayed in vitro for protein synthesis as described in Materials and Methods. To identical reaction aliquots was added m⁷GTP to inhibit initiation, or a buffer control (A). In B, extracts were supplemented with buffer alone, or at five molecules per 80S ribosome of eIF5A(lys), eIF5A(dhp), or FLAG-tagged eIF5A(hyp), the latter protein being purified from yeast. Ribosome concentration in the extract was determined by using the molar extinction coefficient of 80S as 5 × 10⁷ M^-1 cm^-1 at 260 nm and by assuming that ribosomes are 80% of total RNA (25). Experiments shown are representative of multiple independent experiments.