Figure 5.

Gel-retardation assay of glutaminyl-tRNA, EF-Tu, and GTP ternary complex. In A, binding mixtures containing 10 μM of an acid preparation of total tRNA, 10 μM EF-Tu-GTP in 46 mM Tris⋅HCl, pH 7.5, 46 mM KCl, 42 mM NH₄Cl, 6 mM MgCl₂, and 3 mM 2-mercaptoethanol were incubated 5 min at 4°C and fractionated by 6% PAGE. In B, after incubating mixtures as in A, one portion was loaded on the gel being run at 20 V and the other portion was challenged with competitor aminoacyl-tRNA (19 μM final concentration) and incubated another 10 min before loading. The competitor aminoacyl-tRNA was isolated from XAC/A16 cells carrying pGFIB without an amber- suppressor tRNA gene. EF-Tu-GDP was converted to EF-Tu-GTP just before use. Homogeneous preparations of EF-Tu were from either T. aquaticus (A, lanes 1, 2, 5, 6, 9, 10, and B) or T. thermophilus (A, lanes 3, 4, 7, 8, 11, 12). Samples were fractionated at 4°C by 6% PAGE in 25 mM Tris⋅Oac, pH 6.8, 5 mM Mg-Oac, and 5 mM NH₄-Oac, transferred by electroblot and hybridized. The gray input levels were adjusted in photoshop 4.0 from 1.00 to 2.00 in A, lanes 1–4, to better compare them with A, lanes 5–12.