Figure 8

(A) Growth of E. coli expressing Rv1694 in presence of capreomycin: Growth of E. coli expressing Rv1694 in the absence (●) and presence (▼) of capreomycin (100 μmug/ml), and pET28a+ transformed E. coli (○). Error bars represent standard errors from two independent experiments. (B) Inhibition of coupled in vitro transcription-translation by capreomycin: S30 extracts of Rv1694 or pT7Nc transformed E. Coli BL21(DE3) were prepared and in vitro transcription and translation reactions were carried out with either 16 or 80 ng/ml capreomycin. The hemolytic activity of staphylococcal α-hemolysin (used as a reporter) was monitored at OD₅₉₅. All points for all curves were calculated with respect to 100% lysis of rRBCs and average of three independent experiments is shown. Various symbols represent (●) Rv1694 without capreomycin; (○) mock vector without capreomycin; (▼) Rv1694 at 16 ng/ml capreomycin; (∆) mock vector at 16 ng/ml capreomycin; (■) Rv1694 80 at ng/ml capreomycin; (□) mock vector at 80 ng/ml capreomycin. (C) In vitro methylation activity of purified Rv1694 protein on isolated Ribosome: Methylation of E. coli ribosomes in presence or absence of purified Rv1694 and [³H]S-adenosylmethionine. From left, Crossed pattern bar represents the reaction mix with native ribosome ('Nat Rib') and Rv1694 ('Prot'), horizontal pattern bar represents reaction mix with native ribosome only, open bar represents reaction mix with heat (65°C) denatured ribosome ('Den Rib') and Rv1694 and filled bar represents reaction mix with denatured ribosome only. Error bars represent standard errors from triplicate experiments.