TABLE 1.
Aminoacylation properties of in vitro transcribed pseudo-WT and variant human mt tRNALys in the presence of human LysRS
| In vitro transcripts | tRNA position | Wild-type sequence | Mutated sequence | kcat (10−3 sec−1) | KM (μM) | kcat/KM (10−6 sec−1 • nM−1) | Loss |
| Reference molecule | |||||||
| Rz-Ki | 50–64 (TS) | A–U | U •A | 12 | 4.0 | 3.0 | 1 |
| Polymorphism | |||||||
| A8348G | 59 (AccS) | A | G | 11.8 | 2.4 | 4.9 | 0.6 |
| Anticodon triplet mutation | |||||||
| T8324A | 35 (ACL) | U | A | nd | nd | *0.49 10−3 | 6100 |
| Disease-related mutations | |||||||
| A8296G | 2 (AccS) | A–U | G •U | 10.2 | 4.4 | 2.3 | 1.3 |
| G8313A | 24 (DS) | C–G | C •A | nd | nd | *0.43 10−3 | 7000 |
| T8316C | 27 (ACS) | U–A | C • | 2.4 | 1.5 | 1.8 | 2.0 |
| G8328A | 39 (ACS) | C–G | C •A | nd | nd | *0.61 10−3 | 4900 |
| G8342A | 53 (TL) | G–C | A •C | 8.5 | 4.1 | 2.1 | 1.4 |
| A8344G | 55 (TS) | A | G | 14.3 | 1.7 | 8.4 | 0.3 |
| T8356C | 65 (TS) | A–U | A •C | 0.9 | 1.2 | 0.75 | 4.0 |
| T8362G | 71 (AccS) | A–U | A •G | nd | nd | *0.3 | 10 |
| G8363A | 72 (AccS) | C–G | C •A | 22.9 | 10.3 | 2.2 | 1.4 |
Names of the tRNA variants, affected tRNA positions (conventional tRNA numbering, according to Sprinzl et al. 1998), and sequence changes are given in the left part of the table. tRNA structural domains are abbreviated as follows: AccS, acceptor stem; TS, T-stem; DS, D-stem; ACS, anticodon stem; and TL, T-loop. Aminoacylation parameters measured at 25°C in the presence of human cytosolic LysRS are presented on the right. Loss-values (L) are defined as ratio (kcat/KM)Rz-Kl/(kcat/KM)variant. L-values varied by <10%.
*Ratios kcat/KM were determined directly as described (Schulman and Pelka 1988).