Dataset Information

Improved GFP Variants to Study Gene Expression in Haloarchaea.

ABSTRACT: The study of promoter activities in haloarchaea is carried out exclusively using enzymes as reporters. An alternative reporter is the gene encoding the Green Fluorescent Protein (GFP), a simple and fast tool for investigating promoter strengths. However, the GFP variant smRS-GFP, used to analyze protein stabilities in haloarchaea, is not suitable to quantify weak promoter activities, since the fluorescence signal is too low. We enhanced the fluorescence of smRS-GFP 3.3-fold by introducing ten amino acid substitutions, resulting in mGFP6. Using mGFP6 as reporter, we studied six haloarchaeal promoters exhibiting different promoter strengths. The strongest activity was observed with the housekeeping promoters P_fdx of the ferredoxin gene and P2 of the ribosomal 16S rRNA gene. Much lower activities were determined for the promoters of the p-vac region driving the expression of gas vesicle protein (gvp) genes in Halobacterium salinarum PHH1. The basal promoter strength dropped in the order P_pA , P_pO > P_pF , P_pD . All promoters showed a growth-dependent activity pattern. The GvpE-induced activities of P_pA and P_pD were high, but lower compared to the P_fdx or P2 promoter activities. The mGFP6 reporter was also used to investigate the regulatory effects of 5'-untranslated regions (5'-UTRs) of three different gvp mRNAs. A deletion of the 5'-UTR always resulted in an increased expression, implying a negative effect of the 5'-UTRs on translation. Our experiments confirmed mGFP6 as simple, fast and sensitive reporter to study gene expression in haloarchaea.

SUBMITTER: Born J

PROVIDER: S-EPMC6550001 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Improved GFP Variants to Study Gene Expression in Haloarchaea.

Born Johannes J Pfeifer Felicitas F

Frontiers in microbiology 20190529

The study of promoter activities in haloarchaea is carried out exclusively using enzymes as reporters. An alternative reporter is the gene encoding the Green Fluorescent Protein (GFP), a simple and fast tool for investigating promoter strengths. However, the GFP variant smRS-GFP, used to analyze protein stabilities in haloarchaea, is not suitable to quantify weak promoter activities, since the fluorescence signal is too low. We enhanced the fluorescence of smRS-GFP 3.3-fold by introducing ten am ...[more]

PMID: 31191505

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Project description: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.

Dataset Information

Improved GFP Variants to Study Gene Expression in Haloarchaea.

Publications

Improved GFP Variants to Study Gene Expression in Haloarchaea.

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