Table 1.
Binding affinities of wild-type and mutant STAU1 dsRBDs and ARF1 SBS dsRNA
| STAU1 proteina | dsRNAb | K d (nM)c | Affinity factord |
|---|---|---|---|
| dsRBD3/4 | ARF1 SBS (5′-GGCGAGUGCCAGAAGCUGUCUCCUUCGGGAGGCAGUUUCUGGUACUCGCC-3′) | 26.5 ± 2.9 | 1.5 |
| dsRBD3/4 | top 2/3 ARF1 SBS (5′-GGCAGAAGCUGCCUCUUCGGAGGCAGUUUCUGCC-3′) = sARF1 SBS | 17.8 ± 4.3 | 1.0 |
| dsRBD3/4 | bottom 2/3 ARF1 SBS (5′-GGAGUGCCAGAAGCUACGGUUUCUGGUACUCC-3′) | 31.0 ± 0.1 | 1.7 |
| dsRBD3 | sARF1 SBS | 41.4 ± 1.5 | 2.3 |
| dsRBD4 | sARF1 SBS | 74.8 ± 17.6 | 4.3 |
| dsRBD3 I105A S106A | sARF1 SBS | 147.1 ± 35.6 | 3.6 |
| dsRBD3 E110A L113A | sARF1 SBS | 31.5 ± 1.7 | 0.8 |
| dsRBD3 F109A | sARF1 SBS | 98.2 ± 8.7 | 2.4 |
| dsRBD4 Q212A Q215A | sARF1 SBS | 121.2 ± 15.0 | 1.6 |
| dsRBD4 S208A Q212A Q215A | sARF1 SBS | 389.6 ± 38.4 | 5.2 |
| dsRBD3/4 I105A S106A | sARF1 SBS | 28.8 ± 2.7 | 1.6 |
| dsRBD3/4 E110A L113A | sARF1 SBS | 18.5 ± 4.3 | 1.0 |
| dsRBD3/4 I105A S106A E110A L113A | sARF1 SBS | 21.8 ± 6.4 | 1.2 |
| dsRBD3/4 Q212A Q215A | sARF1 SBS | 15.3 ± 0.7 | 0.9 |
| dsRBD3/4 E110A L113A Q212A Q215A | sARF1 SBS | 18.5 ± 1.1 | 1.0 |
| dsRBD3/4 S208A | sARF1 SBS | 28.0 ± 4.4 | 1.6 |
| dsRBD3/4 S208A Q212A Q215A | sARF1 SBS | 87.2 ± 3.0 | 4.9 |
| dsRBD3/4 I105A S106A S208A Q212A Q215A | sARF1 SBS | 43.7 ± 5.2 | 2.5 |
| dsRBD3/4 K133A | sARF1 SBS | 23.2 ± 9.7 | 1.3 |
| dsRBD3/4 R234A R236A | sARF1 SBS | 26.0 ± 9.1 | 1.5 |
| dsRBD3/4 F109A | sARF1 SBS | 29.7 ± 5.8 | 1.7 |
| dsRBD3/4 R209A | sARF1 SBS | 17.7 ± 4.1 | 1.0 |
| dsRBD3/4 F109A R209A | sARF1 SBS | 27.7 ± 5.5 | 1.6 |
aSTAU1 protein construct and mutations are indicated.
bARF1 SBS dsRNA used for FA measurements are indicated.
cAll FA experiments were performed in technical triplicates and the reported Kd values are the average of three experiments with error bars. dAffinity factor for variant RNA or protein relative to top 2/3 ARF1 (sARF1) SBS dsRNA bound to wild-type STAU1 dsRBD3, dsRBD4 or dsRBD3/4, respectively.