Transcriptional processing of an unnatural base pair by eukaryotic RNA polymerase II

Abstract

The development of unnatural base pairs (UBPs) has greatly increased the information storage capacity of DNA, allowing for transcription of unnatural RNA by the heterologously expressed T7 RNA polymerase (RNAP) in Escherichia coli. However, little is known about how UBPs are transcribed by cellular RNA polymerases. Here, we investigate how synthetic unnatural nucleotides, NaM and TPT3, are recognized by eukaryotic RNA polymerase II (Pol II) and found that Pol II is able to selectively recognize UBPs with high fidelity when dTPT3 is in the template strand and rNaMTP acts as the nucleotide substrate. Our structural analysis and molecular dynamics simulation provide structural insights into transcriptional processing of UBPs in a stepwise manner. Intriguingly, we identified a novel 3’-RNA binding site after rNaM addition, termed the swing state. These results may pave the way for future studies to design transcription and translation strategies in higher organisms with expanded genetic codes.

From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma

“…In another approach, with CSR method, Holliger and co-workers evolved a Taq/Tth/Tfl DNAP mutant, 5D4, for the ability of forming and extending other selfpairs of hydrophobic nucleobase analogs, including 5NI and 5NIC 93 . The transcription of some of these UBPs has been demonstrated with several well-studied RNAPs, including T7 RNAP and eukaryotic RNAP II 30,151 suggested that E. coli RNAP could also transcribe DNA containing some of these investigated for the activity of incorporating 2'-modified nucleotides 159 . T7 RNAP mutant Y639F has been found to be able to use various 2'-substituted-NTPs, including dNTPs, 2'-F-dNTPs, and 2'-Am-dNTPs, as substrates during transcription 168,169 , and mutant with one more mutation, T7 RNAP (Y639F, H784A), displayed higher activity against NTPs with bulkier 2'-substitutions, including 2'-OMe and 2'-Az 170 .…”

“…93 The transcription of some of these UBPs has been demonstrated with several well-studied RNAPs, including T7 RNAP and eukaryotic RNAP II. 30,151 Recent in vivo experiments suggested that E . coli RNAP could also transcribe DNA containing some of these UBPs.…”

“…93 The transcription of some of these UBPs has been demonstrated with several well-studied RNAPs, including T7 RNAP and eukaryotic RNAP II. 30,151 Recent in vivo experiments suggested that E. coli RNAP could also transcribe DNA containing some of these UBPs. 152 Recently, reverse transcription of RNA containing TPT3 or NaM has been investigated with several reverse transcriptases or DNAP mutants, including avian myeloblastosis virus (AMV) reverse transcriptase, Moloney murine leukemia virus (MMLV) reverse transcriptase, SuperScript II reverse transcriptase, SuperScript III reverse transcriptase, Super-Script IV reverse transcriptase, and an engineered Taq DNAP with reverse transcription activity, Volcano2G (V2G).…”

Genetic Code Engineering by Natural and Unnatural Base Pair Systems for the Site-Specific Incorporation of Non-Standard Amino Acids Into Proteins

“…This might be because the variable codon performance is the total output of the sequence-dependent translation efficiency, although the recognition of the codon-anticodon interaction might differ in eukaryotic cells ( Zhou et al, 2019 ). They are currently exploring the recognition of the NaM–TPT3 pair by the multi-subunit E. coli RNA polymerase II, as compared to the single-subunit T7 RNA polymerase, to create next generation SSOs ( Hashimoto et al, 2021 ; Oh et al, 2021 ).…”

Efforts toward Further Integration of an Unnatural Base Pair into the Biology of a Semisynthetic Organism

“…12 In vitro kinetic, structural, and simulation studies suggested that eukaryotic multisubunit RNA polymerases recognize the UBP with reasonable efficiency and fidelity when dTPT3 is in the template strand, but less so with dNaM in the template strand. 13 However, it was unclear whether this was also the case with the E. coli enzyme and moreover whether transcription might be altered in an in vivo environment due to for example proofreading, 14,15 transcription−translation coupling, attenuation, or anti-termination mechanisms. 16 Thus, exploring whether the endogenous E. coli RNA polymerase is able to transcribe DNA containing the UBP in the SSO would not only provide a unique means to explore the substrate scope of this important enzyme in vivo but also could eventually provide access to mechanisms that might further optimize the SSO.…”