Evaluation of GFP reporter utility for analysis of transcriptional slippage during gene expression

Ewa Wons , Dawid Kościelniak , Monika Szadkowska , Marian Sęktas

Abstract

Background: Epimutations arising from transcriptional slippage seem to have more important role in regulating gene expression than earlier though. Since the level and the fidelity of transcription primarily determine the overall efficiency of gene expression, all factors contributing to their decrease should be identified and optimized. Results: To examine the influence of A/T homopolymeric sequences on introduction of erroneous nucleotides by slippage mechanism green fluorescence protein (GFP) reporter was chosen. The in- or out-of-frame gfp gene was fused to upstream fragment with variable number of adenine or thymine stretches resulting in several hybrid GFP proteins with diverse amino acids at N-terminus. Here, by using T7 phage expression system we showed that the intensity of GFP fluorescence mainly depends on the number of the retained natural amino acids. While the lack of serine ( S2) residue results in negligible effects, the lack of serine and lysine ( S2K3) contributed to a significant reduction in fluorescence by 2.7-fold for polyA-based in-frame controls and twofold for polyTs. What is more, N-terminal tails amino acid composition was rather of secondary importance, since the whole-cell fluorescence differed in a range of 9–18% between corresponding polyA- and polyT-based constructs. Conclusions: Here we present experimental evidence for utility of GFP reporter for accurate estimation of A/T homopolymeric sequence contribution in transcriptional slippage induction. We showed that the intensity of GFP hybrid fluorescence mainly depends on the number of retained natural amino acids, thus fluorescence raw data need to be referred to appropriate positive control. Moreover, only in case of GFP hybrids with relatively short N-terminal tags the fluorescence level solely reflects production yield, what further indicates the impact of an individual slippage sequence. Our results demonstrate that in contrast to the E. coli enzyme, T7 RNA polymerase exhibits extremely high propensity to slippage even on runs as short as 3 adenine or 4 thymine residues.
Author Ewa Wons (FB / DM)
Ewa Wons,,
- Department of Microbiology
, Dawid Kościelniak (FB / DM)
Dawid Kościelniak,,
- Department of Microbiology
, Monika Szadkowska (FB / DM)
Monika Szadkowska,,
- Department of Microbiology
, Marian Sęktas (FB / DM)
Marian Sęktas,,
- Department of Microbiology
Journal seriesMicrobial Cell Factories, ISSN , e-ISSN 1475-2859, (A 35 pkt)
Issue year2018
Vol17
Pages1-10
Publication size in sheets0.5
Keywords in Englishgreen fluorescent protein reporter, transcriptional slippage, A/T homopolymers, T7 RNA polymerase, E. coli RNA polymerase
DOIDOI:10.1186/s12934-018-0999-3
URL https://doi.org/10.1186/s12934-018-0999-3
Languageen angielski
LicenseJournal (articles only); published final; Uznanie Autorstwa (CC-BY); with publication
Score (nominal)40
ScoreMinisterial score = 35.0, 24-10-2018, ArticleFromJournal
Ministerial score (2013-2016) = 40.0, 24-10-2018, ArticleFromJournal
Publication indicators WoS Impact Factor: 2016 = 3.681 (2) - 2016=4.185 (5)
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