Table 1.
Functional properties of tRNA mutants
| Change to starting tRNA | Suppression efficiency, % | Specificity Gln, % | Glutaminyl-tRNA, % | Relative GlnRS |
|---|---|---|---|---|
| None | 0.2 | 96 | 23 | 1.0 |
| Acceptor stem mutations | ||||
| A1 | 0.6 | 95 | 89 | 1.0 |
| U72 | 4.5 | 97 | 66 | 1.0 |
| A2 | 3.2 | 96 | 62 | 1.0 |
| U4 | 0.8 | 92 | 45 | 0.5 |
| U5 | 0.6 | 96 | 47 | 0.5 |
| G5⋅U68* | 0.2 | 96 | 28 | 0.5 |
| A68* | <0.2 | ND | 17 | ND |
| D stem and loop mutations | ||||
| U11 | 8.0 | Unstable | 70 | 0.5 |
| U13 | 2.9 | 95 | 74 | 0.5 |
| A15 | 2.1 | 94 | 44 | 2.0 |
| U48 | 1.4 | 96 | 41 | 0.5 |
| Anticodon stem and loop mutations | ||||
| G27⋅U43* | 1.0 | 94 | 13 | 0.5 |
| G27⋅C43* | 1.1 | 95 | 16 | 1.0 |
| A31⋅U39* | 6.6 | 93 | 23 | 1.0 |
| C32* | 3.9 | 93 | 54 | 1.0 |
| C32-A38* | 24 | 95 | 49 | 1.0 |
| U38* | <0.2 | ND | 17 | 1.0 |
The gene for the starting tRNA was constructed by directed mutagenesis of an E. coli tRNAAla (UGC anticodon) gene in plasmid pGFIB so that the transcribed molecule would contain C34, U35, A36, C70, and G73. Hydroxylamine and directed mutagenesis were used to isolate active mutants forming blue colonies on X-gal indicator plates (Luria–Bertani agar containing ampicillin and X-gal). Suppression efficiency values are the percentage of the wild-type lacI-Z40 fusion, which averaged 138 units. The value for XAC/A16 cells without suppressor tRNA gene was <0.01%. The amino acid specificity is for residue 3 of dihydrofolate reductase protein, except that residue 10 was analyzed for mutant U4; residue 10 also was analyzed for the starting tRNA, which gave results comparable to residue 3 reported above. The U11 mutant was unstable and its protein could not be analyzed. For Northern blot analysis of glutaminyl-tRNA, mutants were analyzed an average of 3.5 times (average SD ±3.1%). The glutaminyl-tRNA values are calculated as [100 × (aminoacyl-tRNA)/(aminoacyl-tRNA + uncharged tRNA)]. For Western blot analysis of GlnRS, mutants were analyzed an average of 3.1 times (reproducibility of ± 2-fold). The values reported are dilution end-points relative to that of the starting tRNA.
*Mutant constructed by directed mutagenesis.