The synthetic DNA templates encoding the amino acid sequences for Rst1, Rst2, ErmBL and YrbA open reading frames (ORFs) were generated by polymerase chain reaction (PCR) and AccuPrime Taq DNA Polymerase (Thermo Fisher Scientific, USA). The sequences of the primers used for PCR are shown in Supplementary Table S1 . The toe-printing analysis of drug-dependent ribosome stalling analysis (Figure 1D , E ; Figure S1 ) was carried out using Rst1, Rst2, ErmBL and YrbA mRNA templates as previously described (15 (link),16 (link)) with minor modifications. Toe-printing reactions were carried out in 5-μl aliquots containing a PURExpress transcription-translation coupled system (New England Biolabs, USA) to which the test template was added (17 (link)). The reactions were incubated at 37°C for 20–25 min. Reverse transcription on the templates was carried out using radioactively labeled primer NV1 (Supplementary Table S1 ). Primer extension products were resolved on 6% sequencing gels as described previously (18 (link)). The final concentrations of drugs were: 50 μM THR, 50 μM apidaecin (API), 50 μM retapamulin (RET), and 50 μM erythromycin (ERY).
Purexpress transcription translation coupled system
Manufactured by New England Biolabs
Sourced in United States
The PURExpress transcription-translation coupled system is a cell-free protein synthesis kit that allows for the in vitro expression of proteins. The system uses purified components from Escherichia coli to facilitate the coupled transcription and translation of user-supplied DNA templates.
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Lab products found in correlation
3 protocols using purexpress transcription translation coupled system
1
Ribosome Stalling Analysis with Drug Treatments
2
Toe-printing Analysis of Drug-dependent Ribosome Stalling
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The toe-printing analysis of drug-dependent ribosome stalling was carried out using synthetic ermDL DNA template as previously described55 (link)–57 (link) with minor modifications. Toe-printing reactions were carried out in 5-µl aliquots containing PURExpress transcription-translation coupled system (New England Biolabs, USA) to which the DNA template was added58 (link). The reactions were incubated at 37 °C for 20–25 min. Reverse transcription on the templates was carried out using radioactively labeled primer NV1 (5’-GGTTATAATGAATTTTGCTTATTAAC-3’). Primer extension products were resolved on 6% denaturing sequencing gels. The final concentrations of drugs were: 100 µM HygA and 50 µM ERY. In all reactions, we used 50 µM mupirocin (inhibitor of isoleucyl-tRNA-synthetase) to arrest ribosomes at the Ile codon downstream of the macrolide arrest site (Supplementary Fig. 1 ).
3
Profiling Drug-Dependent Translation Stalling
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The synthetic DNA template encoding the amino acid sequence MLILTRRVGETLMIGDEVTVTV (SI Appendix, Table S2 ) was initially generated by polymerase chain reaction (PCR) using E. coli BW25113 genomic DNA and AccuPrime Taq DNA Polymerase (Thermo Fisher Scientific). The sequences of the primers used for PCR are shown in SI Appendix, Table S3 .
The toe-printing assay for drug-dependent translation stalling was performed as previously described in ref. 30 (link) with minor changes. Toe-printing reactions were carried out in 5-µL aliquots containing PURExpress transcription-translation coupled system (New England Biolabs) with 0.5 picomoles of the DNA template added (27 (link)). The reactions were incubated at 37 °C for 15 min. Reverse transcription on the templates was carried out using radioactively labeled primer NV1 (SI Appendix, Table S3 ). Primer extension products were resolved on 6% sequencing gels as described previously (31 (link)). The final concentrations of SAR varied for different experiments.
The toe-printing assay for drug-dependent translation stalling was performed as previously described in ref. 30 (link) with minor changes. Toe-printing reactions were carried out in 5-µL aliquots containing PURExpress transcription-translation coupled system (New England Biolabs) with 0.5 picomoles of the DNA template added (27 (link)). The reactions were incubated at 37 °C for 15 min. Reverse transcription on the templates was carried out using radioactively labeled primer NV1 (
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