Figure 6.

N binds directly to the small ribosomal subunit. 40S small ribosomal subunits were prepared by incubation of ribosomes in the presence of puromycin and purified from large ribosomal subunits and mRNA. (A) Purified 40S subunits were then resedimented on a sucrose gradient. (B) N protein was expressed by in vitro translation in the presence of ³⁵S-methionine, purified from the translation mixture by denaturation with urea, recovery on Ni-NTA beads, renaturation by dialysis and sedimented in parallel with 40S subunits. (C) N was incubated with excess purified 40S subunits prior to sedimentation. Leftward fractions correspond to those from the bottom of the gradient.