FIGURE 3.

Domain IV mutations cause mRNA translocation defects. A fluorescence quenching assay (Studer et al. 2003) was used to measure mRNA translocation complex containing a 3′-pyrene-labeled mRNA. (A,B) A pretranslocation complex was rapidly mixed with mutant forms of EF-G·GTP and quenching of fluorescence of the pyrene label was measured in a stopped-flow fluorimeter. Data were fit to double-exponential curves (Table 1). (C,D) Rates of mRNA quenching for EF-G mutants (C) S587P and (D) S588P were measured manually, due to their low translocation rates. Data could be fit to single-exponential curves.