Table 1.
Regulation of expression and ribosome-binding activity of variant btuB RNAs
| RNA sequence* | β-Galactosidase activity†
|
Primer extension products, %‡
|
||||||
|---|---|---|---|---|---|---|---|---|
| TRX fusion
|
TRL fusion
|
T256
|
A202
|
|||||
| Ado-Cbl− | Ado-Cbl+ | Ado-Cbl− | Ado-Cbl+ | Ado-Cbl− | Ado-Cbl+ | Ado-Cbl− | Ado-Cbl+ | |
| lac | 86.2 | 85.3 (1.0) | ||||||
| btuB wild type | 2,200 | 340 (6.5) | 405 | 3 | 32.5 | 14.1 (2.3) | 4.6 | 21.0 |
| Δ(B12 box) | 100 | 90 (1.1) | 3 | 4 | 10.7 | 9.9 (1.1) | <2 | <2 |
| Hairpin-2 | ||||||||
| M26 | 2,700 | 2400 (1.1) | 590 | 430 | 75.9 | 74.3 (1.0) | 2.4 | 11.6 |
| M27 | 2,870 | 2470 (1.1) | 720 | 560 | 58.7 | 31.9 (1.8) | 2.9 | 16.9 |
| M26.27 | 1,610 | 315 (5.1) | 220 | 17 | 53.8 | 25.8 (2.1) | 2.8 | 14.6 |
| Hairpin-1 | ||||||||
| M31 | 530 | 280 (1.9) | 26 | 5 | 16.3 | 11.5 (1.4) | 1.5 | 1.5 |
| M35 | 390 | 310 (1.3) | 22 | 3 | 13.5 | 13.3 (1.0) | 1.5 | 1.7 |
| M31.35 | 1,600 | 550 (2.9) | 95 | 1 | 28.4 | 11.8 (2.4) | 2.1 | 10.3 |
| M36 | 330 | 390 (0.8) | 13 | 6 | ND | ND | ||
| M31.36 | 650 | 420 (1.5) | 15 | 12 | ND | ND | ||
The sequence changes in the variant btuB leader regions are presented in Fig. 4.
β-Galactosidase activity was measured in cells carrying the variant btuB leader sequences present in btuB-lacZ transcriptional fusions expressed in plasmid pRS415 (TRX fusion) or in btuB-lacZ translational fusions expressed in plasmid pRS414 (TRL fusion). The insert in both plasmids carried the btuB sequences from positions −60 to +450. Cells were grown in the absence and presence of 5 μM Ado-Cbl, as indicated, and the repression ratio (−Ado-Cbl/+Ado-Cbl) is shown in parentheses for the transcriptional fusions.
The levels of the T256 and A202 products are presented as a percentage of the amounts of the major primer extension products. The results are average values of three experiments, one shown in Fig. 5. The value −Ado-Cbl/value +Ado-Cbl is shown in parentheses for the T256 ribosome-binding assay.