Table 1.
The NMR experiments used in the assignment of tRNAAsp, tRNAVal, tRNAPhe and the corresponding acquisition parameters. Unlabeled samples correspond to unmodified and modified tRNAs prepared using unlabeled NTPs or media. 15N-UG samples correspond to in vitro transcribed tRNAs using 15N-labeled GTP and UTP. u-15N samples correspond to uniformly 15N-labeled modified tRNAs produced in 15N-labeled media
| tRNA sample | Experiment | Temp | Field (MHz) | τm (ms) | D1 (s) | NS | Points (F2, F1) | SW (ppm) | AQ (ms) | Carrier frequencies (ppm) | |
|---|---|---|---|---|---|---|---|---|---|---|---|
|
unmodified tRNAAsp |
unlabeled 0.55 mM |
(1H,1H)-NOESY | 311 K | 700 | 150 | 1.0 | 256 | 4096, 744 | 27.9 (1H), 21.6 (1H) |
105 (1H), 24.6 (1H) |
4.7 (1H), 4.7 (1H) |
|
15N-UG 0.27 mM |
(1H,15N)-BEST-TROSY | 311 K | 700 | - | 0.2 | 32 | 4096, 128 | 25.9 (1H), 26 (15N) | 113 (1H), 34.7 (15N) | 4.7 (1H), 154.5 (15N) | |
| modified tRNAAsp |
u-15N 0.43 mM |
(1H,1H)-NOESY | 311 K | 700 | 150 | 1.0 | 296 | 4096, 744 | 27.9 (1H), 21.6 (1H) |
105 (1H), 24.6 (1H) |
4.7 (1H), 4.7 (1H) |
| (1H,15N)-BEST-TROSY | 311 K | 700 | - | 0.2 | 196 | 4096, 256 | 25.9 (1H), 26 (15N) | 113 (1H), 69.4 (15N) | 4.7 (1H), 154.5 (15N) | ||
| (1H,15N)-BEST-TROSY-HNN-COSY | 311 K | 700 | - | 0.3 | 512 | 3072, 324 | 25.9 (1H), 118 (15N) | 84.8 (1H), 19.3 (15N) | 4.7 (1H), 185 (15N) | ||
| (1H,15N)-HSQC-CPMG-NOESY | 311 K | 700 | 130 | 1.1 | 200 | 2714, 404 | 25.9 (1H), 110 (15N) | 74.9 (1H), 25.8 (15N) | 4.7 (1H), 116 (15N) | ||
| (1H,15N)-HSQC | 311 K | 700 | - | 1.0 | 180 | 2048, 280 | 18 (1H), 40 (15N) | 81.1 (1H), 49.3 (15N) |
4.7 (1H), 87 (15N) |
||
|
unmodified tRNAPhe |
unlabeled 1.0 mM |
(1H,1H)-NOESY | 311 K | 950 | 200 | 1.0 | 128 | 5120, 760 | 23.9 (1H), 20 (1H) | 113 (1H), 20 (1H) |
4.7 (1H), 4.7 (1H) |
|
15N-UG 0.45 mM |
(1H,15N)-BEST-TROSY | 311 K | 950 | - | 0.2 | 36 | 4096, 220 | 28.2 (1H), 26 (15N) | 76.4 (1H), 43.9 (15N) | 4.7 (1H), 154.5 (15N) | |
|
modified tRNAPhe |
unlabeled 0.53 mM |
(1H,1H)-NOESY | 311 K | 950 | 200 | 1.0 | 128 | 5120, 760 | 23.9 (1H), 20 (1H) | 113 (1H), 20 (1H) |
4.7 (1H), 4.7 (1H) |
|
u-15N 0.02 mM |
(1H,15N)-BEST-TROSY | 311 K | 950 | - | 0.2 | 212 | 4096, 144 | 28.2 (1H), 26 (15N) | 76.4 (1H), 28.8 (15N) | 4.7 (1H), 154.5 (15N) | |
| (1H,15N)-HSQC | 311 K | 700 | - | 1.0 | 128 | 3072, 168 |
25 (1H), 26 (15N) |
87.8 (1H), 45.5 (15N) | 4.7 (1H), 154.5 (15N) | ||
|
unmodified tRNAVal |
unlabeled 0.32 mM | (1H,1H)-NOESY | 298 K | 700 | 150 | 1.0 | 256 | 4096, 744 | 27.9 (1H), 21.6 (1H) |
105 (1H), 24.6 (1H) |
4.7 (1H), 4.7 (1H) |
|
15N-UG 0.2 mM |
(1H,15N)-BEST-TROSY | 298 K | 700 | - | 0.2 | 40 | 4096, 128 | 25.9 (1H), 26 (15N) | 113 (1H), 34.7 (15N) | 4.7 (1H), 154.5 (15N) | |
|
modified tRNAVal |
unlabeled 1.25 mM | (1H,1H)-NOESY | 298 K | 700 | 150 | 1.0 | 120 | 4096, 568 | 27.9 (1H), 21.6 (1H) |
105 (1H), 18.8 (1H) |
4.7 (1H), 4.7 (1H) |
| (1H,15N)-BEST-TROSY | 298 K | 700 | - | 0.2 | 9000 |
4096, 72 |
25.9 (1H), 26 (15N) |
113 (1H), 19.5 (15N) |
4.7 (1H), 154.5 (15N) | ||