TABLE 2.

Properties of the primary and secondary RNA binding sites of the Rho* mutants

WT and Rho* mutants	Dissociation constants for poly(dC)34 (Kd)a	Km for rC25 oligob	ATPase activity on λtR1 RNAc
	μm		nmol of ATP/min/μg of Rho
WT Rho	9.7 ± 2.0	467.6 ± 13	1.2 ± 0.06
P235H Rho	6.1 ± 0.3	26.7 ± 3	4.2 ± 0.2
R221C Rho	8.4 ± 2	32.8 ± 5	5.3 ± 0.5
I168V Rho	11.7 ± 1.5	52.3 ± 19	2.7 ± 0.4
R221A Rho	11 ± 0.7	62.5 ± 6	9.2 ± 0.8

^a Dissociation constants (K_d) were calculated by fitting the binding isotherms to hyperbolic equation that is obtained from the plots of fluorescence intensities of fluorescein-(dC)₃₄ oligonucleotide at 520 nm upon addition of WT and Rho* mutants. Error bars were obtained from three independent measurements.

^b K_m was calculated from Lineweaver-Burk plot of the form, (1/V) = (K_m + [S])/(V_max[S]), where, V and S are reaction velocity (nanomoles of ATP hydrolyzed/μg of Rho/min) and substrate concentrations, respectively. Intercept on the x axis gave the measure of K_m. Errors were obtained from three measurements.

^c Rates of ATP hydrolysis were calculated from the time-course of ATP hydrolysis by WT and different Rho* mutant using λT_R1 RNA as an inducer. Release of P_i from [γ−³²P]ATP was analyzed on the TLC plates. Error bars were obtained from three measurements.