E. coli NusG was prepared from E. colistrain BL21 Star (DE3) (ThermoFisher) transformed with plasmid pIA247 (encodes
E. coli NusG with C-terminal hexahistidine tag)41 , as described41 . The product (purity
>95%) was stored in aliquots in RNAP storage buffer at
−80°C.
E. coli NusG-N (NusG1-123) was prepared from
E. coli strain BL21 Star (DE3) (ThermoFisher) transformed
with plasmid pRM431-NusG-N [constructed by site-directed mutagenesis of plasmid
pRM431 (encodes E. coli NusG with N-terminal hexahistidine
tag)23 (link), using Vent DNA
polymerase (New England Biolabs) and primers
5’-CAGCAGGTTGGTGATAAGCCGCGTTAGAAAACGCTGTTTGAACCGGGTGAAATGG-3’ and
5’-CCATTTCACCCGGTTCAAACAGCGTTTTCTAACGCGGCTTATCACCAACCTGCTG-3’] as
described for NusG. The product (purity >95%) was stored in aliquots in
RNAP storage buffer at −80°C.
E. coli NusG with C-terminal hexahistidine tag)41 , as described41 . The product (purity
>95%) was stored in aliquots in RNAP storage buffer at
−80°C.
E. coli NusG-N (NusG1-123) was prepared from
E. coli strain BL21 Star (DE3) (ThermoFisher) transformed
with plasmid pRM431-NusG-N [constructed by site-directed mutagenesis of plasmid
pRM431 (encodes E. coli NusG with N-terminal hexahistidine
tag)23 (link), using Vent DNA
polymerase (New England Biolabs) and primers
5’-CAGCAGGTTGGTGATAAGCCGCGTTAGAAAACGCTGTTTGAACCGGGTGAAATGG-3’ and
5’-CCATTTCACCCGGTTCAAACAGCGTTTTCTAACGCGGCTTATCACCAACCTGCTG-3’] as
described for NusG. The product (purity >95%) was stored in aliquots in
RNAP storage buffer at −80°C.
