Table S1.

Approximate distances (Å) between sites of labeling within the ribosome

Signal/structure	PRE (Classical)	PRE (Hybrid)	INT2 (FA)	POST
tRNA/tRNA	35.7 (∼0.65 FRET)	46.2 (0.2, 0.38 FRET)	39.9 (∼0.65 FRET)	39.9 (∼0.65 FRET/no FRET)
S13/tRNA (A-site acp3U47)	74.7 (0.13 FRET)	75.8 (0.13 FRET)	71.3 (∼0.2 FRET)	54.4 (0.42 FRET)
S13/L5	35.7 (0.75 FRET)	44.3 (0.58 FRET)	53.3 (∼0.50 FRET)	37.1 (0.75 FRET)

The interresidue distances between the various sites of labeling used in this study were determined from previously published structures along the translocation reaction coordinate: PRE (Classical; PDB ID code 4V6N) (14), PRE (Hybrid; PDB ID code 4V7D) (15), INT2 (FA-stabilized intermediate; PDB ID code 4V7B) (16), and POST (PDB ID code 4V5F) (17). The corresponding FRET states assigned to the structural pictures are indicated in parentheses for the specific complexes.