Table 1.
Effects of rRNA variants on RNA tertiary structure stability, L11 binding affinity, and cell growth ratesa
| L11BD RNA 3° structureb | L11-RNA affinitiesc | Relative RNA Stabilityd | Relative growth rates, mutant/wild-typee | ||||
|---|---|---|---|---|---|---|---|
| Mutant | Tm °C | ΔH°F kcal/mol | ΔG°F(37°) kcal/mol | ΔG°L11(0°) kcal/mol | ΔΔG°F(37°) kcal/mol | TA531 | MC1000 |
| Wild-type | 41.5 | −30.5 | −0.4 | −8.8(Eco);−8.7(Bst) | 0 | 1 | 1 |
| U1061A | 65.7 | −57.9 | −4.9 | −8.8(Eco);−9.3(Bst) | −4.4 | 1.1 | 1.0 |
| A1085U | -- | -- | -- | > −7.5(Eco) | 4.6f | 1.0 | ND |
| A1085U/U1061A | 43.3 | −37.2 | −0.74 | −8.6 (Bst) | −0.30 | 1.0 | ND |
| C1072U | -- | -- | -- | > −6.3 (Bst) | 9.9f | Lethal | 0.53 |
| C1072U/U1061A | -- | -- | -- | −7.8 (Bst) | 5.0g | 0.85 | 0.83 |
| A1088U | -- | -- | -- | ND | 4.3f | Lethal | 0.74 |
| A1088U/U1061A | 45.3 | −38.5 | −1.0 | > −6.2 (Bst) | −0.6 | 0.84 | 0.91 |
| AA1090GU | 62.0 | −30.0 | −2.2 | −9.3 (Eco) | −1.8 | 1.0 | ND |
Dashes indicates that values could not be extracted from experimental data; ND means the experiment was not run.
Thermodynamic parameters for RNA tertiary structure unfolding were extracted from melting profiles such as those shown in Figure 2. ΔH° and ΔG° values are for the folding reaction. Values for wild-type, U1061A, A1085U, A1088U, AND AA1090GU RNAs were reported by (14); C1072U/U1061A was reported by (18).
L11 binding affinities were obtained from filter binding assays carried out at 0° C as described in Materials and Methods. “Eco” refers to measurements with the E. coli L11 protein, reported by (45). “Bst” are measurements made with the L11 C-terminal domain from B. stearothermophilus, reported by (38) (wild-type), (18) (C1072U/U1061A), and this work (the remainder). In some cases binding was too weak to measure and only an upper limit on the association constant could be estimated.
Stability of each variant L11BD RNA tertiary structure relative to that of the wild type L11BD RNA was estimated from melting curves and L11 binding affinities as described in the text.
Reference doubling times were 64 ± 5 min (TA531-pNO2680) or 25 ± 5 min (MC1000-pNO2680). In order to account for variability in growth curve measurements, mutant growth rates are reported relative to wild-type growth rates measured in the same experiment. “Lethal” means transformants with the mutant plasmid could not be obtained.
Relative stability estimated from that of the corresponding double mutation by adding 4.9 kcal/mol for the stability contributed by U1061A.
Stability estimated from reduced affinity of L11-C76 relative to wild type RNA, and extrapolation to 37 °C using ΔH°F for wild type RNA (14).