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Background
The major gene product of T7 phage is T7 RNA polymerase (T7 RNAP), which catalyzes RNA synthesis and is widely used to synthesize RNA molecules with synthetic modifications and unnatural base pairs (UBPs). T7 RNA polymerase belongs to the single-subunit dextro-polymerase superfamily, which has a strong structural similarity to the Klenow fragment of E. coli DNAP I and consists of an N-terminal domain (residues 1-325) and a polymerase domain (residues 326-883). The polymerase domain may be divided into thumbs, fingers, and palms, and the gap between the two domains is the binding site for the DNA template.
Figure 1. A structural comparison of the fingers sub-domains of RNAP and the Klenow fragment (Source: Jeruzalmi D, et al. 1998)
T7 RNA polymerase translocates along the DNA template during synthesis of the double-stranded product, opening the downstream double-stranded DNA in a process that is independent of helicase proteins. T7 RNAP-mediated transcription can occur in the phases of initiation, elongation, and termination. During initiation, the N-terminal domain (NTD) of T7 RNAP binds to the promoter sequence to form an unstable initiation complex (IC) that generates short RNA transcripts. When the length of the transcript exceeds 10 nucleotides, the NTD promoter rearrangement releases the DNA promoter region and promotes the formation of a stable, progressive enzyme elongation complex. Transcription terminates at a specific sequence or at the end of a linear DNA template, usually producing full-length RNA, but there are cases where non-template additions occur at the 3-terminal. It should also be noted that T7 RNAP can form a dsRNA ring dsRNA product.
Figure 2. The T7 RNAP in the 7-nt RNA intermediate complex is bound to both promoter and downstream DNA (Source: Steitz TA. 2009)
By-products of T7 RNAP RNA synthesis can be immunostimulatory and thus affect the efficacy and safety of clinical applications. These by-products include oligonucleotides generated during transcription initiation, interrupted RNA products due to termination signals, and 3-terminal extension products due to RNA-dependent RNA polymerase activity. Therefore, further optimization and refinement of T7 RNAP is required to reduce by-products and ensure transcription efficiency.
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References
An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts
Nat Biotechnol
Authors: Dousis A, Ravichandran K, Hobert EM, Moore MJ, Rabideau AE.
In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex purification processes, using methods such as reversed-phase high-performance liquid chromatography, to yield safe and effective mRNA-based medicines. To minimize the need for downstream purification processes, we rationally and computationally engineered a double mutant of T7 RNAP that produces substantially less immunostimulatory RNA during IVT compared with wild-type T7 RNAP. The resulting mutant allows for a simplified production process with similar mRNA potency, lower immunostimulatory content and quicker manufacturing time compared with wild-type T7 RNAP. Herein, we describe the computational design and development of this improved T7 RNAP variant.
Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor
Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Here, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP-DNA base editor in <i>Nicotiana benthamiana</i> transient assays to target a transgene expressing <i>GFP</i> under the control of the T7 promoter and observed C-to-T conversions. We then targeted the T7 promoter-driven <i>acetolactate synthase</i> sequence that had been stably integrated in the rice genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as selection pressure for the evolution of the <i>acetolactate synthase</i> sequence, resulting in the enrichment of herbicide-responsive residues. We then validated these herbicide-responsive regions in the transgenic rice plants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance.
COA
Publication 
Figure 1. A structural comparison of the fingers sub-domains of RNAP and the Klenow fragment
Figure 2. The T7 RNAP in the 7-nt RNA intermediate complex is bound to both promoter and downstream DNA
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