TABLE 1.
EF-G protein | GTP binding affinity without ribosome (Kd or Km) | GTP hydrolysis rate (k)a |
Ribosome translocation rate (k)b | |
---|---|---|---|---|
Without ribosome | With ribosome | |||
μm | s−1 | s−1 | s−1 | |
Wild-type | 6.6 ± 0.9c | (5 ± 1) × 10−4 | 4.6 ± 0.4 | 1.8 ± 0.4 |
F95A | 3.9 ± 0.3d | (56 ± 9) × 10−4 | ND | 0.002 ± 0.001 |
D442A | 2.4 ± 0.2d | (162 ± 7) × 10−4 | 0.56 ± 0.02 | 0.044 ± 0.002 |
M461A | 12 ± 1d | (41 ± 4) × 10−4 | 1.18 ± 0.07 | 0.8 ± 0.1 |
L464A | 14 ± 2c | (15 ± 5) × 10−4 | ND | 0.033 ± 0.007 |
H465A | 19 ± 2c | (11 ± 3) × 10−4 | 0.36 ± 0.09 | 0.9 ± 0.4 |
I468A | 13 ± 1d | (29 ± 3) × 10−4 | ND | 0.046 ± 0.003 |
R472A | 7.3 ± 0.9d | (86 ± 2) × 10−4 | ND | 0.072 ± 0.007 |
a Reactions at 20 °C contained EF-G (0.4 μm), GTP (100 μm), and (when present) vacant E. coli ribosome (0.4 μm). Values represent kcat (GTPs hydrolyzed per EF-G/s) under steady-state conditions at saturating GTP concentration.
b Values are the rate of translocation of fluorescent pyrene-mRNA bound to the ribosome (Fig. 7b and supplemental Fig. 3). EF-G (2.5 μm) and GTP (1 mm) were rapidly mixed with pretranslocational ribosomes (0.25 μm) at 20 °C. Values represent the first-order reaction rate constants under pre-steady-state conditions.
c Kd for mant-GTP (without hydrolysis).
d Km for hydrolysis of mant-GTP.