Nucleic Acids Research, 2010, Vol. 38, No. 22 8225 Figure 4. Effects of MtbTopA on mRNA cleavage activities of Rv1495. (A) mRNA cleavage experiments were performed as described under ‘Materials and Methods’ section. A variant concentration of Rv1495 (0.5–2.0 mM) or MtbTopA (40–120 nM) proteins was incubated with 0.5 mM mRNA substrates in the reactions. The mRNA substrate, products and their sequences were indicated by arrows. Rv0054 was used a negative control. (B) Two mutant proteins of MtbTopA and their interactions with Rv1495. The names given to mutants (NTD and CTD) and their amino acid regions are indicated. Bacterial two-hybrid assays for all mutants were performed as described under ‘Materials and Methods’ section. + represents growth on the screening plate; represents no growth on the screening plate. (C) Effects of N-terminal and C-terminal mutant proteins of MtbTopA on the mRNA cleavage activities of Rv1495. The protein concentrations were indicated on top of the panel. M. tuberculosis Rv1495 might interact with MsmTopA. Further conﬁrmation of the conserved interaction was obtained by conducting a GST pull-down assay. As shown in Figure 5B, GST-Rv1495 and His-tagged MsmTopA proteins were co-puriﬁed. A protein of the predicted size of the His-tagged MsmTopA protein could be readily pulled down by GST-Rv1495, which was conﬁrmed by a further western blotting assay (Figure 5B lower panel, lane 2). GST co-incubated with his-tagged MtbTopA did not produce any speciﬁc bands (Figure 5B, lane 3). For further investigation of the physiological signiﬁ- cance of these in vitro reactions, we examined co-immunoprecipitation of the MsmTopA with Rv1495, which was expressed through a pMind recombinant plasmid in M. smegmatis (29). As shown in Figure 5C, the speciﬁc hybridization signal for the expressed Rv1495 in M. smegmatis cell extracts was detected by anti-Rv1495 antibody (lane 1). Using Protein A beads that were ﬁrst conjugated with antibody raised against MsmTopA, an in vivo physical interaction between MsmTopA and Rv1495 was tested. MsmTopA was shown to associate with Rv1495 in vivo because an obvious and a speciﬁc hybridization signal was detected (Figure 5C, lane 2), although to a lesser extent than the signal for the expressed Rv1495 in M. smegmatis cell