Table 1.
Comparison of in vitro affinity data with in vivo activities for Hfq mutants
| Kd (nM hexamer) | |||||||
|---|---|---|---|---|---|---|---|
| Mutant | DsrAa | RNA-U |
RpoS mRNA 5′ UTR |
A27 | Hfq accum. |
rpoS::lacZ assay |
DsrA t1/2 (min) |
| Wild type | 21 ± 1 | 2,500 ± 200 | 49 ± 1 | 39 ± 1 | Yes | 5.7 ± 0.7 | 55 ± 3b |
| 94 ± 5 | |||||||
| Q8A | 19 ± 1 | 2,400 ± 100 | 27 ± 1 | 24 ± 1 | Yes | 3.2 ± 0.2 | – |
| 48 ± 2 | |||||||
| D9A | 14 ± 1 | 320 ± 40 | 35 ± 2 | 36 ± 1 | Yes | 7 ± 1 | – |
| 56 ± 1 | |||||||
| Y25A | 19 ± 1 | – | 32 ± 1 | 196 ± 3 | Yes | – | – |
| 35 ± 1 | |||||||
| Y25N | 15 ± 1 | – | 24 ± 1 | 229 ± 2 | Yes | 4.5 ± 0.6 | – |
| 37 ± 2 | |||||||
| Y25D | 16 ± 1 | – | 22 ± 1 | 224 ± 2 | Yes | 4.0 ± 0.9 | – |
| 38 ± 1 | |||||||
| I30D | 26 ± 1 | – | 35 ± 1 | 363 ± 3 | Yes | 5.2 ± 0.9 | – |
| 41 ± 2 | |||||||
| D40A | 27 ± 1 | 760 ± 90 | 46 ± 1 | 36 ± 1 | No | 0.8 ± 0.1 | – |
| 88 ± 2 | |||||||
| Q41A | 25 ± 1 | 2,100 ± 100 | 70 ± 2 | 26 ± 1 | Yes | 6.4 ± 0.9 | – |
| 98 ± 4 | |||||||
| F42A | 31 ± 1 | 3,500 ± 300 | 50 ± 1 | 40 ± 1 | Impaired | 2.5 ± 0.2 | – |
| 95 ± 4 | |||||||
| Q53A | – | – | – | – | Yes | 3.0 ± 0.6 | – |
| – | |||||||
| Y55A | 82 ± 4 | >20,000 | 82 ± 3 | 115 ± 2 | Yes | 0.8 ± 0.1 | 29 ± 5 |
| 1,600 ± 100 | |||||||
| Y55W | 47 ± 1 | >10,000 | 45 ± 1 | 69 ± 1 | Yes | 4.5 ± 0.8 | – |
| 121 ± 8 | |||||||
| K56A | 101 ± 2 | >20,000 | 104 ± 3 | 59 ± 1 | Yes | 1.0 ± 0.2 | 0.9 ± 0.1c |
| 224 ± 9 | |||||||
| H57A | 44 ± 1 | >20,000 | 53 ± 1 | 55 ± 1 | Yes | 5.4 ± 0.5 | – |
| 108 ± 3 | |||||||
| I59A | – | – | – | – | Yes | 4.2 ± 0.5 | – |
| – | |||||||
| S60A | – | – | – | – | Yes | 5.0 ± 0.3 | – |
| – | |||||||
| Y25D Y55A | 194 ± 9d | >20,000 | 206 ± 6 | 270 ± 12 | – | – | – |
| Y25D Y55W | 34 ± 1 | >10,000 | 54 ± 1 | 400 ± 4 | – | – | – |
| 106 ± 7 | |||||||
| Y25D K56A | 84 ± 3 | >60,000 | 77 ± 2 | 177 ± 3 | – | – | – |
| 320 ± 24 | |||||||
| I30D Y55A | 190 ± 34d | >20,000 | 270 ± 22 | 590 ± 11 | – | – | – |
| I30D Y55W | 41 ± 12 | >20,000 | 55 ± 1 | 312 ± 9 | – | – | – |
| 110 ± 13 | |||||||
| I30D K56A | 52 ± 1 | >60,000 | 71 ± 2 | 280 ± 10 | – | – | – |
| 190 ± 19 | |||||||
DsrA exhibits two sequential transitions—a shift and a supershift. The first value corresponds to K1 and the second value to the supershift, K2. Close inspection of the rpoS mRNA 5′-UTR shifts reveals two apparent shifted species similar to those seen with DsrA. However, the two transitions are sufficiently coupled that they cannot be fit separately.
Hfq− strains have a DsrA t1/2 of 5 ± 2 min.
These data show a biphasic time dependence. A total of 90% of the DsrA RNA decays with a t1/2 of 0.9 min. The final 10% of the RNA shows a much longer t1/2, −35 min.
No clear distinction between K1 and K2 behavior was possible owing to abnormal smearing of the bands. Data were fit to a single transition.