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

Abstract

BackgroundEpimutations 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.ResultsTo 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.ConclusionsHere 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.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0999-3) contains supplementary material, which is available to authorized users.

Non-programmed transcriptional frameshifting is common and highly RNA polymerase type-dependent

“…Each tested fusion gene contained slippery sequence attached upstream to the out-of-frame gfp reporter (− 1 or + 1). These fusions did not appear to have an intracistronic transcription polarity effect and enabled direct measurement of the transcriptional slippage ability [ 52 ]. All nucleotide sequence changes were created to obtain variants of runs with successively increased number of A or T nucleotides (Additional file 1 : Table S3).…”

“…The concentration of the arabinose inductor was chosen to balance the sevenfold faster transcriptional rate of T7 RNAP [ 66 ]. Each time the obtained fluorescence levels of parallel cell lineages were referred to appropriate in-frame controls on either pET24a or pBAD24 backbone, and containing the same number of natural amino acids residues [ 52 ]. Our results (Fig.…”

“…To study the influence of nucleotide type on induction of slippage, several gfp - 1 and gfp + 1 reporter genes were constructed (Additional file 1 : Figure S7) varying in two nucleotides located directly upstream to the slippery-prone sequences: gfpA 5 and gfpT 5 , respectively. Results for the tested di-nucleotides pairs were normalized to appropriate in-frame controls [ 52 ] and compared to the ones obtained for T 2 A 5 ∓1 and A 2 T 5 ∓1, respectively. In the case of A 2 T 5 − 1 template sequence, replacing the 5′-AA with CA, AC and GG did not affect slippage, while we observed 25% increase when AG and CC pairs were analyzed.…”

“…Fluorescence from gfp -less cells was taken as background and subtracted from all values. Strains containing in-frame gfpA 6 0 and gfpT 6 0 fusions were used as positive controls and were taken as 100% [ 52 ].…”

Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures

“…GFP) and some of its derivatives such as enhanced GFP ( abbr. eGFP), have been shown to be useful biosensors for the detection of variations in gene expression both in single-cells and in total populations (Argueta et al 2004 ; Blokpoel et al 2003 ; Cao and Kuipers 2018 ; Carroll et al 2003 ; Chudakov et al 2010 ; Morschhäuser et al 1998 ; Utratna and O'Byrne 2014 ; Wons et al 2018 ). In our previous work (Boy et al 2020 ), a plasmid expression level monitoring method based on the expression of a plasmid-encoded eGFP biosensor has been designed.…”