Table 1.
Competition |
Dual-label |
|||
---|---|---|---|---|
RNAa | Krel | ΔΔG (kcal/mol) | Krel | 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 ± 0.2c | –0.5 ± 0.1c | 0.8 ± 0.3 | 0.2 ± 0.3 |
5WJ_nt6 | 0.8 ± 0.6 | 0.1 (−0.4, +0.9) | 0.4 ± 0.1c | 0.6 ± 0.1c |
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’). Krel = Kd Competitor/Kd 5′domain, where Kd 5′domain = 5.5 ± 2.6 nM, with free energies calculated from ΔΔG = –RTlnKrel 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.