TABLE 2.
Protein | RNAb | K1/2 (without NTP) | K1/2c (with ADP) | K1/2c (with AMP-PNP) |
---|---|---|---|---|
Wild type | R1 | 180 ± 50 | 210 ± 30 | NDd |
Wild type | RNA01 | 230 ± 20 | 270 ± 30 | 35 ± 12 |
Wild type | RNA02 | 200 ± 50 | 290 ± 60 | 40 ± 14 |
F405A | R1 | 890 ± 180 | 1000 ± 200 | 860 ± 200 |
F405A | RNA01 | ND | ND | 700 ± 70 |
F405L | R1 | 250 ± 30 | 220 ± 40 | 120 ± 20 |
F405M | R1 | ND | ND | 53 ± 5 |
The K1/2s (half saturation values) were derived from the best fit of the data to the equation θ = nK[L]m/(1 + K[L]m), where θ is the fraction of RNA bound, n is the number of binding sites, [L] is the ligand concentration, K is the binding constant, and m is the Hill coefficient. Best fits were obtained with either n = 1 or n as a variable (always approximately 1). A minimum of 10% error was assumed in the protein concentrations; larger errors are the standard deviations from the means of three or more independent experiments.
RNA substrates were 44-mer R1 (5′ GGGCGAAUUCAAAACAAAACAAAACUAGCACCGUAAAGCAAGCU 3′), 25-mer RNA01 (5′ UCAUACUUUUCUUUUCUUUUCCAUC 3′), or 25-mer RNA02 (5′ GAUGGAAAAGAAAAGAAAAGUAUGA 3′).
ADP and AMP-PNP concentrations were 5 mM.
ND, not determined.