Purification of Biotinylated RNAP and Recombinant Elongation Factors

Biotinylated RNAP was purified from E. coli BL21DE3 cells transformed with pIA497 plasmid expressing RNAP β’ subunit fused with biotin carboxyl carrier protein, as described previously (Metelev et al., 2017 (link); Sabantsev et al., 2022 (link)). Recombinant GreA and GreB proteins carrying C-terminal 6xHis tag were expressed in E. coli Rosetta cells (NEB) transformed with pJL-GreA-CPH and pJL-GreB-CPH plasmids, respectively, and purified by Ni-chelating HisTrap HP (Cytivia) and gel-filtration Superdex 75 10/30 (Cytivia) column chromatography as described (Borukhov and Goldfarb, 1996 (link)).

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Arseniev A., Panfilov M., Pobegalov G., Potyseva A., Pavlinova P., Yakunina M., Lee J., Borukhov S., Severinov K, & Khodorkovskii M. (2023). Single-molecule studies reveal the off-pathway elemental pause state as a target of streptolydigin inhibition of RNA polymerase and its dramatic enhancement by Gre factors. bioRxiv.

Publication Preprint 2023

HisTrap HP

Biotin carboxyl carrier protein Cells Chromatography E coli Gel filtration Plasmids Proteins Subunit

Corresponding Organization : Rowan University

Other organizations : Rutgers, The State University of New Jersey, Institute of Gene Biology

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Variable analysis

independent variables

Biotinylated RNAP expressing RNAP β' subunit fused with biotin carboxyl carrier protein
Recombinant GreA and GreB proteins carrying C-terminal 6xHis tag

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive control: Not specified
Negative control: Not specified

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