A single mutation attenuates both the transcription termination and RNA-dependent RNA polymerase activity of T7 RNA polymerase

Abstract

Transcription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) was known to response to two types of termination signals, while the mechanism underlying such termination especially the specific elements of the polymerase involved in is still unclear, due to the lack of a termination complex structure. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation S43Y that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNA polymerase (RdRp) of T7 RNAP without affecting the major DNA-dependent RNA polymerase (DdRp) activity of the enzyme, indicating the relationship between transcription termination and RdRp activity, and suggesting a model in which the stem-loop terminator induces the RdRp activity which competes with the ongoing DdRp activity to cause transcription termination. The T7 RNAP S43Y mutant as an enzymatic reagent for in vitro transcription reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.

Exploiting spatial dimensions to enable parallelized continuous directed evolution

“…Reporter plasmids were identical to SC101 accessory plasmids except for the replacement of gIII by gfp . T7 RNAP selection phage (Esvelt et al , 2011; Wu et al , 2021) was constructed by replacing all but the last 202 bp of gIII with the gene encoding T7 RNAP in M13K07 (NEB) helper phage and then removing the p15a origin of replication and aph gene to restore the M13 origin of replication. The mutagenesis plasmid (Esvelt et al , 2011; Badran & Liu, 2015) contained dnaQ926 , dam , and seqA under control of psp operon.…”

FADS and semi-rational design modified T7 RNA polymerase reduced dsRNA production, with lower terminal transferase and RDRP activities

“…It believed to stabilize the non-template DNA and enhance the processivity of T7 RNAP, thus reducing incomplete RNA. Since mutations at 43 and 47 have already been proven to be beneficial for reducing dsRNA [36,37] , we finally created ten single-site (A70, K180, D189, S228, V237, G238, T243, A383, I743, and L747) saturated libraries and employed the traditional microtiter plate method for screening. The selection of a saturated library aims to avoid potential functional impairments of site-directed mutagenesis caused by the limitations of simulation calculations.…”

“…This suggests a clear indication of reduced RDRP activity in the variants, as they show a decreased tendency to utilize RNA as a template. Similarly, this may have had an impact on the terminal transferase activity of T7 RNAP [37] . In addition, we calculated the energy during the extension process of T7 RNAP.…”

“…Simultaneously, helix C (residues 28-71) and subdomain H (residues 151-190) undergo a process of refolding. Helix C2 (residues 46-55) stacks onto the helix C1 (residues [28][29][30][31][32][33][34][35][36][37][38][39][40][41], resulting in the fusion of the original two short helices into a continuous long helix. Additionally, the loop (residues 173-186) within subdomain H transforms into an α-helix.…”

An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts

“…During IVT, an RNAP is responsible for the transcription of RNA from a DNA template. Although IVT is a recognized process in research laboratories, optimization for industrial-scale production has only recently begun [4][5][6][7][8][9][10] .…”