Non-programmed transcriptional frameshifting is common and highly RNA polymerase type-dependent
Dawid Kościelniak , Ewa Wons , Karolina Wilkowska , Marian Sęktas
AbstractBackground: The viral or host systems for a gene expression assume repeatability of the process and high quality of the protein product. Since level and fidelity of transcription primarily determines the overall efficiency, all factors contributing to their decrease should be identified and optimized. Among many observed processes, non-programmed insertion/deletion (indel) of nucleotide during transcription (slippage) occurring at homopolymeric A/T sequences within a gene can considerably impact its expression. To date, no comparative study of the most utilized Escherichia coli and T7 bacteriophage RNA polymerases (RNAP) propensity for this type of erroneous mRNA synthesis has been reported. To address this issue we evaluated the influence of shift-prone A/T sequences by assessing indel-dependent phenotypic changes. RNAP-specific expression profile was examined using two of the most potent promoters, ParaBAD of E. coli and φ10 of phage T7. Results: Here we report on the first systematic study on requirements for efficient transcriptional slippage by T7 phage and cellular RNAPs considering three parameters: homopolymer length, template type, and frameshift directionality preferences. Using a series of out-of-frame gfp reporter genes fused to a variety of A/T homopolymeric sequences we show that T7 RNAP has an exceptional potential for generating frameshifts and is capable of slipping on as few as three adenine or four thymidine residues in a row, in a flanking sequence-dependent manner. In contrast, bacterial RNAP exhibits a relatively low ability to baypass indel mutations and requires a run of at least 7 tymidine and even more adenine residues. This difference comes from involvement of various intrinsic proofreading properties. Our studies demonstrate distinct preference towards a specific homopolymer in slippage induction. Whereas insertion slippage performed by T7 RNAP (but not deletion) occurs tendentiously on poly(A) rather than on poly(T) runs, strong bias towards poly(T) for the host RNAP is observed. Conclusions: Intrinsic RNAP slippage properties involve trade-offs between accuracy, speed and processivity of transcription. Viral T7 RNAP manifests far greater inclinations to the transcriptional slippage than E. coli RNAP. This possibly plays an important role in driving bacteriophage adaptation and therefore could be considered as beneficial. However, from biotechnological and experimental viewpoint, this might create some problems, and strongly argues for employing bacterial expression systems, stocked with proofreading mechanisms.
|Journal series||Microbial Cell Factories, ISSN 1475-2859, (A 40 pkt)|
|Publication size in sheets||0.75|
|Keywords in English||Recombinant proteins, E. coli RNAP, T7 bacteriophage RNAP, expression system, GFP reporter, transcriptional slippage, indel errors|
|ASJC Classification||; ;|
|License||Journal (articles only); published final; ; with publication|
|Score||= 40.0, 04-02-2020, ArticleFromJournal|
|Publication indicators||= 0; : 2016 = 1.228; : 2018 = 4.402 (2) - 2018=4.669 (5)|
* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.