T7 RNA Polymerase

T7 RNA polymerase (T7RNAP) can transcribe the unnatural base pair (UBP), Ds–Pa, which is hydrophobic and lacks hydrogen bonds. Through kinetics, crystallography, and mutagenesis, T7RNAP was shown to incorporate, with selectivity and low efficiency, the cognates unnatural triphosphates (DsTP or PaTP) opposite the template Ds or Pa.
Structural studies reveal that both Ds and Pa are bound at the pre-insertion site, not by hydrogen bonds but through unique stacking and metal-mediated interactions. DsTP is in a primed state that is close to the active site, which explains the fast kinetics of its incorporation. In contrast, PaTP must undergo a large conformational change. Mutation of M635 completely abolishes UBP transcription without affecting natural substrate incorporation, demonstrating a key role for this residue in UBP recognition.

Fig. 1 Unnatural template loading by T7 RNA polymerase. (Oh J.; et al. 2023)

Wang Q et al. developed a targeted mutagenesis system, named CgMutaT7, for the continuous in vivo evolution of target proteins in the industrial workhorse Corynebacterium glutamicum. This system is based on a fusion of T7 RNA polymerase with cytosine deaminase and a uracil-DNA glycosylase inhibitor, connected by linker peptides. The fusion protein induces mutations specifically at the targeted DNA sequence, which is transcribed by T7 RNA polymerase from the T7 promoter. After optimization, the resulting CgMutaT74 system was highly efficient. They applied this system to the continuous evolution of xylose isomerase with CgMutaT74 and obtained a strain that metabolizes xylose more efficiently.

Fig. 2 Targeted continuous evolution system via T7-guided base editing. (Wang Q.; et al. 2024)