Table 1. Open in new tab Relative...

	Competition	Dual-label
16S rRNA	1.6 ± 0.8	–0.3 ± 0.4	N.D.^b
RNA(ΔH6-14)	1.1 ± 0.4	–0.1 ± 0.2	0.5 ± 0.1	0.5 ± 0.1
RNA(ΔH5-14)	2.3 ± 0.2^c	–0.5 ± 0.1^c	0.8 ± 0.3	0.2 ± 0.3
5WJ_nt6	0.8 ± 0.6	0.1 (−0.4, +0.9)	0.4 ± 0.1^c	0.6 ± 0.1^c
5WJ	0.4 ± 0.1	0.7 ± 0.2	0.6 ± 0.1	0.5 ± 0.2
5WJ:H18trunc	N.D.^d		0.9 ± 0.1	0.1 ± 0.1
5WJ:BstH17	N.D.^d		0.8 ± 0.4	0.2 (–0.3,+0.5)
5WJ:TthH17	N.D.^d		5.8 ± 1.8	–1.1 ± 0.2
5WJ:C526A	N.D.^d		0.5 ± 0.2	0.4 ± 0.3

	Competition	Dual-label
16S rRNA	1.6 ± 0.8	–0.3 ± 0.4	N.D.^b
RNA(ΔH6-14)	1.1 ± 0.4	–0.1 ± 0.2	0.5 ± 0.1	0.5 ± 0.1
RNA(ΔH5-14)	2.3 ± 0.2^c	–0.5 ± 0.1^c	0.8 ± 0.3	0.2 ± 0.3
5WJ_nt6	0.8 ± 0.6	0.1 (−0.4, +0.9)	0.4 ± 0.1^c	0.6 ± 0.1^c
5WJ	0.4 ± 0.1	0.7 ± 0.2	0.6 ± 0.1	0.5 ± 0.2
5WJ:H18trunc	N.D.^d		0.9 ± 0.1	0.1 ± 0.1
5WJ:BstH17	N.D.^d		0.8 ± 0.4	0.2 (–0.3,+0.5)
5WJ:TthH17	N.D.^d		5.8 ± 1.8	–1.1 ± 0.2
5WJ:C526A	N.D.^d		0.5 ± 0.2	0.4 ± 0.3

Table 1.

Relative dissociation constants and free energies for S4 binding measured by competition against 5′ domain RNA

	Competition		Dual-label
RNA^a	K_rel	ΔΔG (kcal/mol)	K_rel	ΔΔG (kcal/mol)
16S rRNA	1.6 ± 0.8	–0.3 ± 0.4	N.D.^b
RNA(ΔH6-14)	1.1 ± 0.4	–0.1 ± 0.2	0.5 ± 0.1	0.5 ± 0.1
RNA(ΔH5-14)	2.3 ± 0.2^c	–0.5 ± 0.1^c	0.8 ± 0.3	0.2 ± 0.3
5WJ_nt6	0.8 ± 0.6	0.1 (−0.4, +0.9)	0.4 ± 0.1^c	0.6 ± 0.1^c
5WJ	0.4 ± 0.1	0.7 ± 0.2	0.6 ± 0.1	0.5 ± 0.2
5WJ:H18trunc	N.D.^d		0.9 ± 0.1	0.1 ± 0.1
5WJ:BstH17	N.D.^d		0.8 ± 0.4	0.2 (–0.3,+0.5)
5WJ:TthH17	N.D.^d		5.8 ± 1.8	–1.1 ± 0.2
5WJ:C526A	N.D.^d		0.5 ± 0.2	0.4 ± 0.3

	Competition		Dual-label
RNA^a	K_rel	ΔΔG (kcal/mol)	K_rel	ΔΔG (kcal/mol)
16S rRNA	1.6 ± 0.8	–0.3 ± 0.4	N.D.^b
RNA(ΔH6-14)	1.1 ± 0.4	–0.1 ± 0.2	0.5 ± 0.1	0.5 ± 0.1
RNA(ΔH5-14)	2.3 ± 0.2^c	–0.5 ± 0.1^c	0.8 ± 0.3	0.2 ± 0.3
5WJ_nt6	0.8 ± 0.6	0.1 (−0.4, +0.9)	0.4 ± 0.1^c	0.6 ± 0.1^c
5WJ	0.4 ± 0.1	0.7 ± 0.2	0.6 ± 0.1	0.5 ± 0.2
5WJ:H18trunc	N.D.^d		0.9 ± 0.1	0.1 ± 0.1
5WJ:BstH17	N.D.^d		0.8 ± 0.4	0.2 (–0.3,+0.5)
5WJ:TthH17	N.D.^d		5.8 ± 1.8	–1.1 ± 0.2
5WJ:C526A	N.D.^d		0.5 ± 0.2	0.4 ± 0.3

^aBinding affinity of S4 for competitor RNA was measured relative to ³²P-labeled 5′domain RNA at 42°C in HKM4 buffer, as described in ‘Materials and methods’ section. Data were fit to Equation (1) (‘Competition’) or Equation (2) (‘Dual-label’). K_rel = K_{d Competitor}/K_{d 5′domain}, where K_{d 5′domain} = 5.5 ± 2.6 nM, with free energies calculated from ΔΔG = –RTlnK_rel at 315.15 K. The relative affinities and dissociation free energy values were averaged over three or more experiments for ‘Competition’, and from three to seven reactions for ‘Dual-label’ competitions, unless stated otherwise.

^b16S rRNA and 16S–S4 complexes were poorly resolved.

^cValues reported from two experiments.

^dThese RNAs were tested against 5′ domain RNA only through dual-label competition.

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