Table 4. The effect of altering the sequence of the D and TΨC loops.
| Aab9T | pre-tRNAGly | |||
|---|---|---|---|---|
| kreact (min−1) | KM(sto) (nM) | kreact (min−1) | KM(sto) (nM) | |
| wild-type | 0.42 ± 0.01 | 40 ± 6 | 1.67 ± 0.03 | 4.8 ± 0.4 |
| G18→A18 | 1.87 ± 0.07 | 22 ± 3 | ||
| G19→A19, C56→U56a | 1.78 ± 0.06 | 7.7 ± 1.2 | ||
| U54→C54 | 0.017 ± 0.001 | 88 ± 12 | ||
| U55→C55 | 0.159 ± 0.005 | 80 ± 9 | ||
| C56→U56 | 0.13 ± 0.01 | 208 ± 27 | 1.81 ± 0.05 | 6.4 ± 0.9 |
| A57→G57 | 0.47 ± 0.02 | 34 ± 6 | ||
| A57→C57 | 0.018 ± 0.001 | 462 ± 63 | 1.56 ± 0.05 | 6.7 ± 0.9 |
| A57→U57 | 0.009 ± 0.001 | 144 ± 52 | ||
| A58→G58 | 0.060 ± 0.001 | 108 ± 7 | ||
| TCCUUUUAb | 0.017 ± 0.001 | 72 ± 18 | ||
Single-turnover kinetic constants of PRORP3 for the processing of minimal-substrate (Aab9T) and pre-tRNAGly variants with different identity of conserved nucleotides in the D and/or TΨC loops (see Figure 2A and B for position within the secondary structures; best-fit values ± standard error from the fitting of at least three replicate experiments each).
aThe identity of C56 was varied in order to maintain tertiary Watson–Crick base pairing with nucleotide 19.
bThe sequence of the wild-type TΨC loop of Aab9T and pre-tRNAGly is UUCAAGU.