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. 2018 Mar 15;137:11-19.
doi: 10.1016/j.ymeth.2017.12.003. Epub 2017 Dec 30.

The Cell Free Protein Synthesis System From the Model Filamentous Fungus Neurospora Crassa

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The Cell Free Protein Synthesis System From the Model Filamentous Fungus Neurospora Crassa

Cheng Wu et al. Methods. .
Free PMC article

Abstract

Cell-free protein synthesis (CFPS) can be used in many applications to produce polypeptides and to analyze mechanisms of mRNA translation. Here we describe how to make and use a CPFS system from the model filamentous fungus Neurospora crassa. The extensive genetic resources available in this system provide capacities to exploit robust CFPS for understanding translational control. Included are procedures for the growth and harvesting of cells, the preparation of cell-free extracts that serve as the source of the translational machinery in the CFPS and the preparation of synthetic mRNA to program the CFPS. Methods to accomplish cell-free translation and analyze protein synthesis, and to map positions of ribosomes on mRNAs by toeprinting, are described.

Figures

Fig. 1
Fig. 1
AAP-mediated regulation of mRNA translation in response to Arg in the N. crassa CFPS examined by measurement of luciferase reporter activity. Triplicate reactions were programmed with the indicated mRNAs and luciferase activity levels measured after 30 min of translation as described in the text. Reactions contained low Arg (black) or high Arg (gray). Amounts of luciferase produced, normalized to the amount produced from the control fLuc mRNA (lacking any known 5′-regulatory sequences) in low Arg, are shown. Plasmids used [–29] to generate the mRNAs were: fLuc, pPQ101 (originally called pHLucNFS); AAPw, pPR301 (wild- type AAP as uORF); AAPm, pPS301 (non-regulatory D12N mutant AAP as uORF); AAPw Fusion, pKL105 (wild-type AAP fused in frame with luciferase); AAPm Fusion, pKLS105 (D12N mutant AAP fused in frame with luciferase). Error bars represent SD values.
Fig. 2
Fig. 2
AAP-mediated regulation of mRNA translation in response to Arg in the N. crassa CFPS examined by toeprinting analysis of ribosome positions on mRNA. N.crassa CFPS was programmed with equal amounts (60 ng) of AAPw mRNA that contains the wild type AAP as a uORF upstream of luciferase. Cycloheximide (Cyh) was either not added to translation reactions (−), or was added either prior to starting the translation reactions (T0) or after 15 min of incubation (T15). Radiola-beled primer ZW4 [27] was used for primer extension analysis and for sequencing the pPR301 DNA template. The nucleotide complementary to the dideoxynu-cleotide added to each sequencing reaction is indicated below the corresponding lane to enable the sequence of the template to be directly deduced. Arrowhead, toeprints corresponding to ribosomes stalled at the termination codon of AAP; open circle, toeprints corresponding to ribosomes at AAP initiation codon; closed circle, toeprints corresponding to ribosomes stalled at the luciferase initiation codon. Boxes in the sequencing marker lanes (top to bottom) indicate AAP initiation, AAP termination, and luciferase initiation codons, respectively. –RNA, extract without RNA;-EXT, RNA without extract.

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