In vitro transcription-translation assay.

A New England Biolabs Inc. PureExpress delta ribosome kit was used for the assay. A T7 promoter driven nano-luc reporter was used as the template (40). The reactions were set up as per the manufacturers protocol. To determine the concentration of CLY required to inhibit 50% ribosome activity, 20 nM purified M. smegmatis 70s ribosomes was incubated with 1, 2, 5, 10, 20, or 50 nM CLY and kept at room temperature for 15 min. Four microliters of solution A, 1.2 μl of Factor mix from the kit, and 50 ng of DNA template were then added to the reaction mixture and incubated at 37°C for 1 h. After 1 h, 2 μl from the reaction mixture was mixed with 18 μl of water, 20 μl of luciferase assay buffer, and 0.4 μl of luciferin substrate and incubated for 5 min at room temperature. The luminescence generated from the luciferase was measured in a Veritas microplate luminometer (Turner Biosystems). To determine the effect of Ms5102 on clindamycin inhibition, 20 nM purified M. smegmatis 70s ribosomes was incubated with 200 nM purified Ms5102 for 15 min at room temperature, followed by the addition of 2 nM clindamycin for an additional 10 min. The amount of luciferase activity in each sample was determined as described above.