An Overview of T7 RNA Polymerase

T7 RNA Polymerase Promoter

The essential genes of T7 linear genome can be divided into three categories, which are expressed at different stages of T7 infection cycle. Class I genes create favorable conditions for phage growth by being expressed early in infection, class II genes are mainly involved in encoding DNA replication proteins, and class III genes are expressed late in phage growth, encoding mainly structural gene products. In the early stage of infection, class I genes are transcribed by host (E. coli) RNA polymerase, while II and III genes are transcribed by T7 RNAP.

The transcriptional complex needs to be formed at the beginning of transcription, and the reaction can be broken down into four steps: (1) initial promoter recognition and binding, (2) promoter melting, (3) RNA extension to the length of polymerase release promoter (/ 9nT), and (4) RNA extension to make the extension complex completely stable (/ 14nT). 23bp T7 promoter plays a role in the first three steps of this process, which is consistent with the general principle of macromolecular tissue in polymerase. It shows a three-part modular structure, in which three basically non-overlapping sequence elements participate in three different functions: recognition, melting and effective initial transcription.

• Promoter Recognition

The common sequences of the most active class III T7 promoters include the upstream 17bp sequence of the transcriptional initiation site and the downstream 6bp sequence. Sequence-specific recognition involves sequences between -5 and -17. Gene mutations in this region affect promoter activity and affinity for polymerase. Despite its specificity, the binding of T7 RNAP to its promoter was surprisingly weak, and it is speculated that this relatively weak binding reflects the polymerase's need to release the promoter at the end of the initial reaction so that it can move down the DNA as an extension complex.

Figure 2. Structure of a class III T7 promoter with the functions of different parts of the promoter indicated.
(Source: Sousa R, et al. 2003)

• Promoter Melting

Whereas mutations in the -5 to -17 region affect promoter and polymerase affinity, mutations downstream of -5 reduce promoter activity without affecting affinity for polymerase. In fact, while promoters must be double-stranded upstream of -5 to have full activity and affinity, T7 promoters that are single-stranded downstream of -5 (lacking non-template chains) are active, forming a more stable initiation complex with a higher affinity for polymerase than full double-stranded promoters.

Studies have shown that -1 to -4 sequences are conserved, not because of their base-specific interaction with polymerase, but because they promote the melting of the promoter, and in the transcriptional initiation reaction, the -5 to -17 parts of the promoter remain base paired, while the DNA downstream of -5 is melted.

• Transcription Initiation

The formation of the first phosphodiester bond must first bind the two initiating NTPs. The consensus is that both are GTP on T7 promoter, although GDP or GMP can be replaced at +1 position, and +2 NTP must have triphosphate moiety.

The existence of cytosine in the +1 and +2 positions of the template chain of T7 promoter is very important, it can overcome the normal space limitation and directly select the starting site of transcription. If the template bases of +1 and +2 are not cytosine, but cytosine appears at +3 and +4, then the polymerase can start with +3.

• Initial Transcription

The formation of the first phosphodiester bond does not complete the transcriptional initiation process. Instead, the polymerase enters a stage called "initial" or "abortive" transcription. At this stage, short RNA (2-9 nucleotides in length) is often released from the transcriptional complex, then the polymerase is reactivated and usually does not dissociate from the promoter.

Application Limitation of T7 RNA Polymerase

T7 RNA polymerase is widely used in RNA synthesis in vitro and protein expression in vivo (bacterial high expression system). However, in addition to the advantages of high transcriptional efficiency, strong extension ability and high fidelity, it also has some disadvantages that cannot be ignored as a tool for RNA synthesis in vitro. T7 RNAP may produce many by-products during the synthesis of RNA, including oligonucleotides produced during transcriptional initiation, interrupted RNA products caused by termination signals, and 3'- terminal extension products caused by RNA-dependent RNA polymerase activity. These immunostimulatory double-stranded RNA transcribed from product templates may affect efficacy and safety, especially in therapeutic applications. Therefore, we urgently need to optimize and improve T7 RNAP so that it cannot only maintain efficient transcription, but also reduce the problem of heterogeneity of RNA products.

Current research shows that by combining structural, mechanical, computational and laboratory screening methods to design mutated T7 RNAP, the production of dsRNA by-products can also be significantly reduced. Researchers designed a double mutant of T7 RNAP through reasonable calculation. Compared with wild-type T7 RNAP, it produces much less immunostimulatory RNA in the in vitro transcription process, can simplify the production process, has similar mRNA potency, lower immunostimulatory content and faster production time.

And it is reported that a single mutation (S43Y) significantly reduced the termination efficiency of T7 RNAP on all tested transcriptional terminators. Coincidentally, the S43Y mutation almost eliminated the RNA-dependent RNAP (RdRp) activity of T7 RNAP without hindering the main DNA-dependent RNAP (DdRp) activity of the enzyme, thus reducing the unwanted termination of run-off RNA synthesis and producing RNA with higher terminal uniformity.