TABLE 1.

Dissociation constants for binding of RNA species to ABH8 domains

See supplemental Methods and the legend to Fig. 5 for buffer conditions.

Protein constructs	RNA molecules
	tRNA-Glya	tRNA-Glua	17-mer stem-loopb	Aptamer ABH8-2.2b	Control 17-merb
1–133	3.0 ± 0.9 μm	830 ± 330 nm	1.3 ± 0.4 μm	3.9 ± 0.2 μm	NDc
1–354	2.9 ± 0.8 μm	2.3 ± 1.8 μm	1.4 ± 0.2 μm	2.3 ± 0.4 μmd	6.3 ± 1.0 μm
1–354(C341A/C349A)	ND	ND	808 ± 151 nm	2 ± 0.2 μm	ND
25–354-His6	ND	ND	26 ± 5 μm	4.8 ± 0.3 μm	ND
352–663+Trm112e	ND	ND	9.1 ± 0.9 μm	61 ± 6 μm	ND
1–663+Trm112e	490 ± 290 nm	830 ± 590 nm	240 ± 29 nm	240 ± 50 nmd	350 ± 20 nm

^a Values calculated from radiolabeled filter binding assays at room temperature.

^b Values calculated from fluorescence anisotropy assays at 25 °C.

^c ND, not determined.

^d Filter binding assays on the same RNA species without a fluorescent label gave equivalent binding affinity for the 1–663+Trm112 construct (350 ± 20 nm) but higher affinity for the 1–354 construct (290 ± 90 nm). Either the fluorescent label reduces aptamer affinity for the 1–354 construct or a conformational change in this construct upon filter binding increases its binding affinity.

^e Assays on constructs containing the MTase domain (352–663) displayed reduced binding at the highest protein concentrations, suggesting aggregation, limiting the accuracy of the dissociation constants measured for these constructs.