Table 1. Affinity of wild-type and mutated S7 for its mRNA.
| Mutation |
K′a (µM–1) |
Relative affinity for str mRNA |
Relative affinity for 16S rRNAa |
| None |
6.8 ± 0.9 |
1.00 |
1.00 |
| Δ1–17 |
0.6 ± 0.1 |
0.09 |
n.d. |
| Δ72–89 |
1.9 ± 0.3 |
0.28 |
0.34 |
| Δ138–178 |
3.7 ± 0.4 |
0.54 |
0.58 |
| Δ148–178 |
6.6 ± 1.2 |
0.97 |
1.06 |
| R3Q |
2.9 ± 1.1 |
0.43 |
0.57 |
| Q8A |
1.8 ± 0.3 |
0.26 |
0.21 |
| F17G |
1.3 ± 0.2 |
0.19 |
0.17 |
| K34Q |
6.7 ± 0.9 |
0.99 |
1.04 |
| K35Q |
1.9 ± 0.3 |
0.28 |
0.45 |
| G54S |
3.3 ± 0.8 |
0.49 |
0.40 |
| Y84A |
4.7 ± 0.6 |
0.69 |
0.64 |
| K113Q |
4.0 ± 0.7 |
0.59 |
0.42 |
| M115G |
0.7 ± 0.1 |
0.10 |
0.26 |
| K136Q |
6.0 ± 0.8 |
0.88 |
0.98 |
| R142Q |
5.6 ± 0.9 |
0.82 |
0.83 |
| M143A | 4.4 ± 0.8 | 0.65 | 0.68 |
A fragment of the str operon mRNA encompassing the intercistronic region between S12 and S7 coding sequences was synthesized in vitro and incubated with increasing amounts of protein. The binding affinity of wild-type and mutated S7 for the str mRNA fragment was measured by a nitrocellulose filter binding assay. Binding assays were performed in a high-ionic-strength buffer [20 mM Mg(OAc)2, 350 mM KOAc]. K′a values are means ± standard deviation of at least three independent experiments; n.d., not detectable.
aThe relative affinity of the S7 mutants for a fragment corresponding to the lower half of the 3′ major domain of 16S rRNA was determined by Robert et al. (5) in a high-ionic-strength buffer comparable to that used in this study (20 mM MgCl2, 300 mM KCl).