Table 2.
Aminoacylation and in vivo binding of M. barkeri tRNAPyl variants by M. barkeri PylRS
| tRNA | Mutations | KM, μM | kcat, s−1 | kcat/KM | L | Binding, % |
|---|---|---|---|---|---|---|
| tRNAPyl | Wild type | 0.23 ± 0.04 | (4.0 ± 1.2) × 10−2 | 1.74 × 10−1 | 1 | 100 |
| tRNAPyl | G73A | 0.48 ± 0.14 | (1.5 ± 0.2) × 10−3 | 3.00 × 10−3 | 58.0 | 55 |
| tRNAPyl | C72U | 0.10 ± 0.03 | (1.6 ± 0.3) × 10−4 | 1.60 × 10−3 | 108.7 | 76 |
| tRNAPyl | C13:G22→A:U | 0.16 ± 0.04 | (1.9 ± 0.3) × 10−2 | 1.16 × 10−1 | 1.5 | 95 |
| tRNAPyl | A45G | 0.51 ± 0.08 | (3.2 ± 0.9) × 10−2 | 6.31 × 10−2 | 2.8 | 92 |
| tRNAPyl | G48U | 0.39 ± 0.04 | (9.2 ± 3.1) × 10−3 | 2.36 × 10−2 | 7.4 | 79 |
| tRNAPyl | G51:C63→U:A | 0.97 ± 0.23 | (8.2 ± 1.4) × 10−3 | 8.49 × 10−3 | 20.5 | 69 |
| tRNAPyl | U33G | 0.12 ± 0.02 | (1.2 ± 0.2) × 10−3 | 1.00 × 10−2 | 17.4 | 67 |
| tRNAPyl | A37C | 0.20 ± 0.04 | (1.3 ± 0.3) × 10−3 | 6.63 × 10−3 | 26.2 | 55 |
| tRNAPyl | Anticodon CUA→CAA | 0.09 ± 0.02 | (2.0 ± 0.4) × 10−2 | 2.19 × 10−1 | 0.8 | 82 |
| tRNAPyl | Anticodon CUA→AUA | 0.67 ± 0.10 | (1.7 ± 0.4) × 10−2 | 2.57 × 10−2 | 6.8 | 94 |
| tRNAPyl | Anticodon CUA→CUU | 0.17 ± 0.02 | (3.2 ± 0.6) × 10−2 | 1.88 × 10−1 | 0.9 | 86 |
| tRNASer | Wild type | ND | ND | ND | ND | ND |
| tRNASer/Pyl | Chimera 1 | 0.26 ± 0.04 | (2.1 ± 0.4) × 10−2 | 8.15 × 10−3 | 2.13 | 78.3 |
| tRNASer/Pyl | Chimera 2 | 0.29 ± 0.05 | (2.3 ± 0.5) × 10−2 | 7.93 × 10−3 | 2.19 | 82.6 |
In vivo binding of tRNAPyl to PylRS was determined by the yeast three-hybrid method as described in Materials and Methods. L represents loss of aminoacylation efficiency and is calculated as (kcat/KM mutant)/(kcat/KM wild type). ND, the activity could not be detected.