Table 1.
Effect of Mtq2 and Trm112 mutants on eRF1 methylation in vitro
| eRF1 MTase activity (%) | ||
|---|---|---|
| Trm112 mutants | ||
| N43R (D36) | 58 ± 6 | Located in Ec-Trm112-Mtq2 interface |
| F46D (I39) | 0 | |
| R53E (T46) | 0 | |
| I125D (I115) | 0 | |
| A106E (V96) | 59 ± 11 | Outside from Ec-Mtq2-Trm112 interface |
| E107K (E97) | 61 ± 3 | |
| I118E (I108) | 51 ± 8 | |
| Y120E (P110) | 74 ± 3 | |
| N123R (G113) | 58 ± 1 | |
| Mtq2 mutants | ||
| E16K (E5) | 15 ± 3 | Active site |
| Y15F (Y4) | 0 | |
| E19K (E8) | 0 | |
| D20N (D9) | 0 | |
| F22A (Y11) | 0 | |
| D26K/E29K (D15/E18) | 0 | |
| N122A (N85) | 0 | |
| R207A (R149) | 6.5 ± 2.1 | |
| R207E (R149) | <2 | |
| E212K (E154) | 65 ± 6 | |
| D77A (D51) | 0 | SAM binding |
| D106A/L107A (D69/L70) | 0 | |
All these Mtq2 mutants were co-expressed with wild-type Sc-Trm112 in E. coli and purified using standard protocols. With the exception of the F22A mutant, none of these mutations affected significantly complex formation, solubility and CD spectra (data not shown). Although its CD spectrum was comparable to that of wild-type complex, the Mtq2 (F22A)-Trm112 mutant complex was less stable than wild-type complex. The eRF1 MTase activity of each complex is expressed as a percentage of activity obtained with wild-type enzyme. Absolute activities were measured at least in triplicate, as the initial velocity of the reaction (pmol of eRF1 methylated by second). The E. cuniculi numbering is indicated in parenthesis.