Figure 3. HF interacts with both the site for amino acid activation and the site for docking the 3′-end of tRNA.

a, Thermal melting¹⁹ of ProRS in the presence of different ligands. HF binds in the absence of ATP^a, and binds more strongly in the presence of ATP^a. b, HF and the comparator Pro-SA block formation of Pro-AMP, in the proline-dependent ATP-PPi exchange reaction. The inset shows that HF had no effect on the alanine-dependent ATP-PPi exchange reaction with AlaRS. c, HF mobilizes the release of Pro-AMP from ProRS. The ProRS:³H-Pro-AMP complex was prepared on ice and then isolated on a column and for 10 min was left untreated, or exposed to HF, or exposed to Pro-SA, respectively. The complex was then re-run on the column and the amount of bound ³H-Pro-AMP was determined. The inset shows that the isolated ³H-Pro was activated (as ³H-Pro-AMP) and could be transferred to tRNA. Error bars are s.e.m. (n=2).