Practical Thoughts
This is the beginning of a set of resources for thinking practically about transcription.
T7 RNA polymerase, unlike more complex multi-subunit RNA polymerases 1) is highly specific for a relatively small promoter sequence, 2) initiates transcription at a good rate, 3) elongates about 5 times faster, and is readily purified in good yield and purity. All of this makes it an excellent system for synthesizing RNA of all lengths.
Enzymology studies of T7 RNA polymerase have used somewhat physiological concentrations of substrates and magnesium: 0.5 mM each ATP, CTP, UTP, GTP and 15 mM Mg(II), with typically 5 min reaction times.
“High yield” applications of the system use >10X-higher concentrations: up to 7.5 mM each ATP, CTP, UTP, and GTP and 40 mM Mg(II), with 4-12 hr reaction times.
We have a lot to say about the above. Stay tuned to this page…
Why T7 RNA polymerase?
| T7 RNA polymerase | Bacterial RNA polymerase | |
| Optimized for | speed & efficiency | complex regulation |
| Promoter specificity | high specificity for a well-defined, short promoter | requires cofactors for recognizing a variety of complex and long promoters |
| Elongation rate | 250 bases/s | 50 bases/s |
| Motor | very strong motor protein | moderate motor protein |
| Stability | single subunit; very stable; over-expresses easily | multi-subunit + accessory factors |
T3, SP6, K11, Syn 5, et al. are all in the same family of single subunit enzymes. As such, they all share the same beneficial properties outline for T7 RNA polymerase and should work well in applications. T7 RNA polymerase is by far the most well-studied of the group and so holds a slight advantage IMHO. Nevertheless, when you see “T7 RNA polymerase” here think “T7 family of RNA polymerases.”