Table 1.
Histidylation properties of resected RNA pseudoknots and comparison with those of the BMV tRNA-like domain and yeast tRNAHis
| RNAs, duplex no. | Charging level,* apparent % | Km, μM | Vmax, arbitrary units | L† |
|---|---|---|---|---|
| RNA/RNA duplexes | ||||
| Most efficient duplex | ||||
| 1 G117 C4 | 9.3 (9.3) | 0.7 (0.9) | 42.0 (45.6) | 1 (1) |
| Single mutants | ||||
| 2 U117 C4 | 6.7 (7.4) | 1.0 (0.6) | 29.6 (21.5) | 2 (2) |
| 3 A117 C4 | 7.1 (7.2) | 1.4 (0.8) | 26.2 (14.9) | 3 (3) |
| 4 G117 A4 | 10.7 (10.7) | 1.7 (1.7) | 29.6 (29.6) | 3 (3) |
| 5 G117 U4 | 6.2 (6.2) | 25.0 (1.0) | 252.0 (22.3) | 6 (3) |
| 6 C117 C4 | 2.0 (2.1) | 3.6 (1.2) | 6.1 (4.8) | 35 (15) |
| 7 G117 G4 | 4.0 (4.0) | 2.4 (2.4) | 3.6 (3.6) | 40 (40) |
| Double mutants | ||||
| 8 U117 U4 | 3.0 (3.3) | 0.8 (1.9) | 6.7 (7.6) | 7 (15) |
| 9 A117 A4 (wt) | 4.0 (3.3) | 1.8 (1.1) | 6.8 (5.6) | 16 (12) |
| 10 A117 U4 | 2.0 (2.0) | 4.2 (2.0) | 9.7 (5.7) | 26 (21) |
| 11 C117 U4 | 1.1 (1.1) | 1.9 (1.5) | 2.2 (2.2) | 52 (41) |
| 12 U117 G4 | 0.9 (1.3) | 2.3 (123.0) | 1.9 (24.2) | 73 (305) |
| 13 U117 A4 | 1.3 (1.0) | 1.8 (3.1) | 1.3 (1.8) | 83 (103) |
| 14 A117 G4 | 0.7 (1.0) | 3.0 (37.4) | 1.8 (6.4) | 100 (350) |
| 15 C117 A4 | 0.9 (1.0) | 2.2 (9.5) | 1.1 (4.5) | 120 (127) |
| 16 C117 G4 | 0.2 (0.5) | 0.9 (11.8) | 0.2 (0.5) | 270 (1416) |
| BMV tRNA-kike domain | ||||
| 17 A117 A4 (wt) | 40 | 1.3 | 170 | 0.46 |
| yeast tRNAHis transcript | ||||
| 18 G-1 A73 | 100 | 0.3 | 35 103 | 5.1 10−4 |
| 10-mers alone | ||||
| 19–22 A4, C4, G4, or U4 | 0.0 | ND | ND | — |
Experimental data are given for resected pseudoknots with closed and open (between brackets) circles.
Charging levels were obtained for 40 min incubation in the presence of equivalent amounts of enzyme (2.5-fold excess as compared to initial rate conditions for resected pseudoknot charging) and were calculated assuming that all 10-mers are chargeable (all hybridized). For initial rate determinations enzyme was 30 units/ml incubation mixture (except for tRNAHis where it was 150-fold decreased) and incubation times up to 5 min (variants 1–3, 17), 10 min (variants 4, 8–11), 20 min (variants 6, 7, 12–15), and 50 min (variant 16). All aminoacylations were performed at 15°C as described.
Relative losses in catalytic efficiencies of histidine acceptors compared to efficiency of duplex 1 are expressed as L = (Vmax/Km)duplex 1/(Vmax/Km)histidine acceptor. ND, not detectable. The A117/A4 combination found in the BMV tRNA-like structure and other bromovirus RNAs (31) is in bold (duplex 9). Combinations present in RNAs from phylogenetically related cucumo- and bromoviruses (31) are underlined. Duplexes 3, 4 and 13 contain identity elements present in natural substrates of eukaryotic HisRSs (A117/C4 in the tRNA-like domains of the RNAs from tobacco mosaic virus and its satellite virus, G117/A4 in yeast and other eukaryotic tRNAHis species, and U117/A4 in the tRNA-like domain of the RNA from TYMV; for sequence data see refs. 4, 32 and 33).