Table 1.
Observed rate constants for the various human CPEB3 ribozymes
| RNA | Standard assaysa | Co-transcriptional assaysb | ||||
|---|---|---|---|---|---|---|
| kobs (min−1) | Amplitudec | Fold effectd | kobs (min−1) | Amplitudec | Fold effectd | |
| Human −59/68 (WT) |
0.55±0.1 | 21±2% | 55 | ≥0.5e | 17±2% | ≥50 |
| 0.03±0.003 | 54±2% | 3 | 0.05±0.004 | 75±2% | 5 | |
| nr | 26±4% | - | nr | 8±0.7% | - | |
| Human −8/68 (WT) |
0.33±0.08 | 18±2% | 33 | ≥0.5e | 13±2% | ≥50 |
| 0.03±0.005 | 55±4% | 3 | 0.06±0.004 | 79±2% | 6 | |
| nr | 27±5% | - | nr | 8±0.3% | - | |
| Human −8/68 (C–2A) |
2.6±0.5 | 16±1% | 260 | ≥2.5e | 18±1% | ≥250 |
| 0.53±0.06 | 60±5% | 53 | 0.49±0.02 | 80±1% | 49 | |
| nr | 24±5% | - | nr | 2±0.5% | - | |
| Chimp −8/68 (WT) |
2.4±0.6 | 42±3% | 240 | ≥2.2 | 29±3% | ≥220 |
| 0.44±0.02 | 21±2% | 44 | 0.72±0.06 | 63±3% | 72 | |
| nr | 37±4% | - | nr | 8±0.2% | - | |
| Chimp −8/68 (C–2A) |
3.2±0.2 | 37±2% | 320 | ≥2.8 | 34±1% | ≥280 |
| 0.6±0.09 | 17±1% | 60 | 1.1±0.2 | 60±1% | 110 | |
| nr | 45±2% | - | nr | 6±0.5% | - | |
| −89/68/52f | 0.01 | 70% | 1 | - | - | - |
| −30/99 HDV (G11C/U27Δ)g |
63 | 86% | 6,300 | - | - | - |
Each data point is the average of at least two trials ± the standard error of the experiments. All data are for denaturing PAGE fractionation.
“nr”, fraction remaining unreacted at the end of the reaction.
Self-cleavage reactions of full-length RNA, which were initiated by addition of MgCl2 and fit to eq 2. All data are for singly purified, [γ-32P]GTP–labeled RNA.
Co-transcriptional self-cleavage assays, which were fit to eq 3
Amplitudes for RNA associated with each fraction. For standard assays, the amplitude of the nr phase was determined from the fits. Errors for the nr phase were calculated using the square root of the sum of the squares of the other two amplitude errors. For co-transcriptional assays, the amplitude of the nr phase was determined experimentally from the average of long (2-8 h) time points, and associated errors are the standard deviation. These experimentally determined nr values were held constant in fits.
Fold effects are relative to 0.01 min−1 for human −89/68/52 CPEB3 ribozyme (Salehi-Ashtiani et al, Science 2006).
This value was estimated by substituting different values for k1 into eq 3 and finding the lowest value that still gave a good fit to the data using the amplitudes given.
Salehi-Ashtiani et al, Science 2006.
From Brown et al 2004.