Have you ever wondered why T3, T7 and SP6 phage DNA-dependent RNA polymerases are commonly used in in vitro transcription and in coupled transcription/translation reactions? As a follow-up on the article about cell-free protein synthesis, I thought it might be of interest to explain why phage RNA polymerases are frequently used in in vitro reactions in place of prokaryotic or eukaryotic RNA polymerases.
- Phage RNA polymerases are very simple structurally. They are approximately 100KDa in size. Prokaryotic and eukaryotic RNA polymerases in comparison are multi-subunit enzymes (1). During preparation of coupled transcription/translation extract of E. coli for example, the E. coli transcriptases are removed along with the membranes. Adding back the multi-subunit enzyme is cumbersome (2). There is thus a huge advantage and ease in using the monomeric phage RNAP in in vitro extracts.
- The phage enzymes can perform the complete transcription process without the need for additional protein factors (3).
- The recognition sequence (promoter) of these enzymes is short and highly conserved (4). It is therefore easier to have that sequence coded as part of the DNA template.
- Phage polymerases are also extremely specific for their respective promoter sequence (4). Due to this, high level of expression of genes cloned under the control of the respective phage promoter sequence is possible.
- The phage enzymes are extremely fast and highly processive. At 37°C, T7 RNAP can move along the DNA strand at a speed that is 5 to 8 times faster than E. coli RNAP. This allows transcription reaction to proceed at a much faster rate than native E. coli RNAP (2).
The simplicity in structure and function of the phage polymerases has made them an attractive alternative in invtiro reactions.
Do you know of other advantages for the use of these enzymes? Or if you have questions or comments about this post, drop us a line in the comments section to tell us about it. We welcome and value your feedback!
- Chamberlain M et al. 1973. Characterization of T7 specific Ribonucleic Acid Polymerase. Journal of Biological Chemistry. 278 (6), 2235-2211.
- Iskakova, M. et al . (2006) Nucleic Acid Research. 34 (19), e135.
- Tunitskaya, V.L. et al. (2002) Structural-Functional Analysis of Bacteriophage T7 RNA Polymerase. Biochemistry.67 (10) 1124–35
- Cheetham, G.M.T. et al. (2000) Insights into transcription: structure and function of single subunit DNA-dependent RNA-polymerases. Current Opinion in Structural Biology. 10. 117-123