TABLE 1.

Biochemical activities of Fus^R EF-G proteins (derived from wild-type E. coli EF-G)

Values represent the means ± S.D. from three or more separate reactions. ND, not detectable.

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 k_cat (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 K_d for mant-GTP (without hydrolysis).

^d K_m for hydrolysis of mant-GTP.