The gateway pDEST17 expression vector encodes a −1 ribosomal frameshifting sequence

Belfield, Eric J.; Hughes, Richard K.; Tsesmetzis, Nicolas; Naldrett, Mike J.; Casey, R.

doi:10.1093/nar/gkm003

Cited by 5 publications

(5 citation statements)

References 44 publications

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“…Compared to the common 1–5% frameshifting efficiencies, the frameshifting percentages observed here are substantially higher. This is probably due to the lack of the stimulator because similar high efficiencies were reported elsewhere in the absence of stimulators. ,, …”

Section: Resultssupporting

confidence: 72%

“…This is probably due to the lack of the stimulator because similar high efficiencies were reported elsewhere in the absence of stimulators. 24 , 27 , 28 …”

Section: Resultsmentioning

confidence: 99%

“…This is probably due to the lack of the stimulator because similar high efficiencies were reported elsewhere in the absence of stimulators. 24,27,28 Extensive ribosome pausing over the poly(A) sequences was observed that could lead to translational frameshiftings. 24 Meanwhile, "−1", "−4", and "+2" frameshiftings were observed simultaneously on a similar mRNA.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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High-Efficiency “–1” and “–2” Ribosomal Frameshiftings Revealed by Force Spectroscopy

et al. 2017

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Ribosomal frameshifting is a rare but ubiquitous process that is being studied extensively. Meanwhile, frameshifting motifs without any secondary mRNA structures were identified but rarely studied experimentally. We report unambiguous observation of highly efficient “–1” and “–2” frameshiftings on a GA7G slippery mRNA without the downstream secondary structure, using force-induced remnant magnetization spectroscopy combined with unique probing schemes. The result represents the first experimental evidence of multiple frameshifting steps. It is also one of the rare reports of the “–2” frameshifting. Our assay removed the ambiguity of transcriptional slippage involvement in other frameshifting assays. Two significant insights for the frameshifting mechanism were revealed. First, EF-G·GTP is indispensable to frameshifting. Although EFG·GDPCP has been shown to prompt translocation before, we found that it could not induce frameshifting. This implies that the GTP hydrolysis is responsible for the codon–anticodon re-pairing in frameshifting, which corroborates our previous mechanical force measurement of EF-G·GTP. Second, translation in all three reading frames of the slippery sequence can be induced by the corresponding in-frame aminoacyl tRNAs. Although A-site tRNA is known to affect the partition between “0” and “–1” frameshifting, it has not been reported that all three reading frames can be translated by their corresponding tRNAs. The in vitro results were confirmed by toe-printing assay and protein sequencing.

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Section: Resultssupporting

confidence: 72%

“…This is probably due to the lack of the stimulator because similar high efficiencies were reported elsewhere in the absence of stimulators. 24 , 27 , 28 …”

Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

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High-Efficiency “–1” and “–2” Ribosomal Frameshiftings Revealed by Force Spectroscopy

et al. 2017

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“…Poly(A) tracks resemble ribosome “slippery” sequences that have been associated with translational frameshifts ( 20 , 21 ). Recent studies suggest that poly(A) tracks can induce “sliding” of E. coli ribosomes resulting in frameshifting ( 10 , 22 ).…”

mentioning

confidence: 99%

Translational control by lysine-encoding A-rich sequences

Arthur

Pavlovic-Djuranovic

Koutmou³

et al. 2015

Sci. Adv.

107

176

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Regulation of gene expression involves a wide array of cellular mechanisms that control the abundance of the RNA or protein products of that gene. Here we describe a gene-regulatory mechanism that is based on poly(A) tracks that stall the translation apparatus. We show that creating longer or shorter runs of adenosine nucleotides, without changes in the amino acid sequence, alters the protein output and the stability of mRNA. Sometimes these changes result in the production of an alternative “frame-shifted” protein product. These observations are corroborated using reporter constructs and in the context of recombinant gene sequences. Approximately two percent of genes in the human genome may be subject to this uncharacterized, yet fundamental form of gene regulation. The potential pool of regulated genes encodes many proteins involved in nucleic acid binding. We hypothesize that the genes we identify are part of a large network whose expression is fine-tuned by poly(A)-tracks, and we provide a mechanism through which synonymous mutations may influence gene expression in pathological states.

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“…The hGAD65 and hGAD65mut sequences were individually cloned in the Gateway destination vector pDEST17, which allows the induction of transcription with IPTG (Belfield et al 2007 ). A chloramphenicol-resistance gene in the same vector was used as a negative control.…”

Section: Resultsmentioning

confidence: 99%

Comparative analysis of different biofactories for the production of a major diabetes autoantigen

et al. 2013

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The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major diabetes autoantigen that can be used for the diagnosis and (more recently) the treatment of autoimmune diabetes. We previously reported that a catalytically-inactive version (hGAD65mut) accumulated to tenfold higher levels than its active counterpart in transgenic tobacco plants, providing a safe and less expensive source of the protein compared to mammalian production platforms. Here we show that hGAD65mut is also produced at higher levels than hGAD65 by transient expression in Nicotiana benthamiana (using either the pK7WG2 or MagnICON vectors), in insect cells using baculovirus vectors, and in bacterial cells using an inducible-expression system, although the latter system is unsuitable because hGAD65mut accumulates within inclusion bodies. The most productive of these platforms was the MagnICON system, which achieved yields of 78.8 μg/g fresh leaf weight (FLW) but this was substantially less than the best-performing elite transgenic tobacco plants, which reached 114.3 μg/g FLW after six generations of self-crossing. The transgenic system was found to be the most productive and cost-effective although the breeding process took 3 years to complete. The MagnICON system was less productive overall, but generated large amounts of protein in a few days. Both plant-based systems were therefore advantageous over the baculovirus-based production platform in our hands.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-013-9749-9) contains supplementary material, which is available to authorized users.

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The gateway pDEST17 expression vector encodes a −1 ribosomal frameshifting sequence

Cited by 5 publications

References 44 publications

High-Efficiency “–1” and “–2” Ribosomal Frameshiftings Revealed by Force Spectroscopy

High-Efficiency “–1” and “–2” Ribosomal Frameshiftings Revealed by Force Spectroscopy

Translational control by lysine-encoding A-rich sequences

Comparative analysis of different biofactories for the production of a major diabetes autoantigen

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