Table 1.

Influence of Zn²⁺, Sr²⁺ and/or $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ on the binding of ptRNA^Gly to E.coli M1 RNA

Metal ion(s)		Kd (nM)	Average endpoint
Transition Me2+	Other
—	—	10 000 ± 3000	0.66
20 mM Zn2+	—	≥20 000a	n.d.a
20 mM Cd2+	—	2100 ± 400	0.75
—	5 mM Sr2+	240 ± 15	0.93
20 mM Zn2+	5 mM Sr2+	700 ± 300	0.59
—	80 mM Sr2+	4 ± 0.5	0.98
20 mM Zn2+	80 mM Sr2+	9 ± 0.5	0.92
20 mM Zn2+	5 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	2486 ± 400	0.55
—	20 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	751 ± 200	0.75
20 mM Zn2+	20 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	350 ± 120	0.65
—	80 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	79 ± 35	0.26
20 mM Zn2+	80 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	53 ± 6	0.28

Spin column assay for the determination of K_d values were performed at pH 6.0 and 1 M NH₄OAc using trace amounts of 5′-endlabeled ptRNA^Gly; individual K_d values are based on three to six independent experiments and were calculated by non-linear regression analysis (program Grafit, Erithacus Software) using the equation: f_c = f_t · [P RNA]_free/(K_d + [P RNA]_free), where f_c = fraction of ptRNA in the complex, and f_t = maximum fraction of ptRNA that is able to bind to P RNA (endpoint). Endpoints in the right column are the theoretical ones obtained by the fitting procedure; however, theoretical and experimentally measured endpoints were generally in good agreement; average endpoints were normalized to that for 40 mM Sr²⁺ (see Figure 3A).

^aK_d and endpoint values (n.d. = not determined) could not be determined with reasonably low errors owing to very low ribozyme–substrate affinity; the K_d of 20 000 nM in the presence of 20 mM Zn²⁺ alone is a lower limit estimate.