mRNA Decay and RNA‐Degrading Machines in Prokaryotes and Eukaryotes

Carpousis, Agamemnon J.; Dreyfus, Marc

doi:10.1002/3527603433.ch5

Cited by 1 publication

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References 96 publications

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“…We constructed a GFP-mRNA that is flanked by the highly conserved sequences enclosing the 23S rRNA and forms a strong base-paired stem resulting in a pseudo-circularization of the mRNA similar to that of the precursor rRNA (‘stable’ GFP-mRNA, lower panel in Figure 3a ). We expected a prolonged mRNA half-life, since the endo RNase E prefers substrates with unpaired 5′ ends and the exo PNPase and RNase II are specific for single-stranded RNAs ( 32 ). As a control we used the same mRNA except that the mutation in the 5′ flanking region disrupts the complementarity and thus pseudo-circularized mRNA fails to be produced (‘unstable’ GFP-mRNA).…”

Section: Resultsmentioning

confidence: 99%

Troubleshooting coupled in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins

Iskakova

Szaflarski

Dreyfus

et al. 2006

Nucleic Acids Research

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Cell-free coupled transcription–translation systems with bacterial lysates are widely used to synthesize recombinant proteins in amounts of several mg per ml. By using reporter green fluorescence protein (GFP) we demonstrate that proteins are synthesized with an unsatisfyingly low-active fraction of (50 ± 20)%. One reason is probably the T7 polymerase used, being up to eight times faster than the intrinsic transcriptase and thus breaking the coupling between transcription and translation in bacterial systems. The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20°C. A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids. Accordingly, a second addition of amino acids after 10 h during an incubation at 20°C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity.

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Section: Resultsmentioning

confidence: 99%