Table 1.
Promoter specificities of mutant RNAPs
| RNAP* | Plasmid† | Relative promoter utilization‡
|
Relative specific activity§ | |||
|---|---|---|---|---|---|---|
| −8T | −8A | −8C | −8G | |||
| Wild type | pBH117 | 1 | 0.01 | 0.01 | 0.01 | 1 |
| Q758C | pMR74 | 0.15 | 0.43 | 0.06 | 1 | 0.71 |
| Q758S | pMR57 | 0.15 | 1 | 0.01 | 0.86 | 0.53 |
| Q758R | pMR64 | 0.03 | 0.07 | 1 | 0.04 | 0.31 |
| Q758E | pMR75 | 1 | 0.01 | 0.01 | 0.01 | 0.23 |
| Q758N | pMR65 | 0.07 | 0.08 | 0.01 | 1 | 0.16 |
| Q758K | pMR50 | 0.03 | 0.04 | 1 | 0.01 | 0.15 |
| Q758G | pMR55 | 0.33 | 0.73 | 0.16 | 1 | 0.11 |
| Q758A | pMR72 | 0.26 | 0.97 | 0.33 | 1 | 0.08 |
| Q758V | pMR62 | 0.66 | 0.67 | 0.29 | 1 | 0.04 |
| Q758H | pMR71 | 0.30 | 1 | 0.47 | 0.47 | 0.03 |
| Q758L | pMR66 | 1 | 0.49 | 0.31 | 0.96 | 0.03 |
| Q758I | pMR56 | 0.53 | 0.19 | 0.23 | 1 | 0.02 |
| Q758Y | pMR76 | 0.71 | 1 | 0.66 | 0.85 | 0.01 |
| Q758T | pMR70 | 0.99 | 1 | 0.01 | 0.96 | 0.01 |
| Q758D | pMR73 | ND | ND | ND | ND | <0.01 |
| Q758W | pMR63 | ND | ND | ND | ND | <0.01 |
| Q758F | pMR54 | ND | ND | ND | ND | <0.01 |
| Q758P | pMR67 | ND | ND | ND | ND | <0.01 |
| subs(755-761) | pBH212 | ND | ND | ND | ND | <0.01 |
| subs(755-757) | pMR49 | ND | ND | ND | ND | <0.01 |
| subs(758-759) | pBH213 | ND | ND | ND | ND | <0.01 |
| subs(743-777) | pMR106 | ND | ND | ND | ND | <0.01 |
Individual amino acid substitutions at position 758 are identified. In the RNAPs encoded by pBH212, pMR49, pBH213, and pMR106, the amino acid residues in T7 RNAP in the range indicated have been replaced with the corresponding residues from K11 RNAP (see Fig. 1).
The plasmid that encodes the indicated RNAP.
The preference of the RNAP for a promoter having the indicated bp at position −8 was determined as shown in Fig. 3C. The radioactivity in each electrophoretic species was quantified by exposing the gel to a PhosphorImager screen (Molecular Dynamics) taking into account the base composition and sizes of the individual transcripts. The amount of RNA produced from the test promoter relative to that of the reference promoter was determined [(PT7X)/(PT7ref)], and the data in each set were normalized to the strongest promoter in that series (24). For mutant enzymes that had very low activity at any promoter (<0.01 that of the wild-type enzyme), these values were not calculated (ND).
The activity of each mutant enzyme at its strongest promoter relative to the activity of the same amount of WT enzyme at a consensus promoter.