Figure 4.

Measurement of the dissociation constant of LeuRS proteins for tRNA^Leu by gel-shift assay. (A) In vitro transcribed ³²P-labelled A.aeolicus was incubated with increasing concentrations of the β subunits or whole αβ-LeuRS (see Materials and Methods). Apparent dissociation constants (K_d) of the proteins were estimated from quantification of the free and retarded tRNA. Band shifts are displayed along the linear schematic representation of the β subunit (NH₂- to the COOH-end). For clarity the protein concentrations were omitted from each picture. The large increases of K_d were shown by a shift of the gel picture towards the right side, which symbolizes the high values. (B) K_d values and binding energy variations for the β subunits and αβ-LeuRS proteins.