MtbRho has a monomeric size of 65 kDa, as estimated from sequence analysis and also shown by mass spectrometry. However, the protein showed anomalous migration at,80 kDa on SDS-PAGE, probably due to the presence of clusters of polar residues in the subdomain. In the several steps involved in Rho-mediated transcription termination, the first step of Rho’s action is its binding to the rut site. EcRho is known to have a preference for C-rich, unstructured RNA for initial Rho binding and polycytidylic acid has been used to study Rho activity in vitro. The residues of EcRho that have been implicated in binding to a C-rich sequence the motifs involved in RNA-dependent ATPase activity are similar in MtbRho. The ATPase activity of MtbRho in presence of synthetic homopolymeric polyC, polyA and polyU is shown in Figure 1A. While polyC is, not unexpectedly, the best substrate, polyA and polyU also stimulate the hydrolysis of ATP. Homopolymeric polyC, polyA and polyU are, however, not natural substrates of MtbRho. In vivo, MtbRho would function in presence of various mycobacterial RNAs and it is likely that MtbRho has evolved a greater ability to interact with its natural substrates. To assess if MtbRho could use mycobacterial RNA as substrate for ATPase, cellular RNA from M. smegmatis mc2155 was used. The results presented in Figure 1B show that MtbRho can hydrolyse ATP in presence of mycobacterial RNAs. The ATPase activity was specific to MtbRho as it was inhibited by Bicyclomcycin. To study if a specific mycobacterial RNA molecule could be used as a substrate for ATPase activity, we used a RNA corresponding to the region downstream of the sdaA gene of M. tuberculosis genome. This RNA was chosen as in silico analysis revealed the absence of intrinsic terminator downstream of the sdaA gene and hence it is likely target for Rho-dependent termination. The results presented in Figure 2A and B show that MtbRho can hydrolyse ATP in presence of sdaA RNA. But, MtbRho is inherently a weaker ATPase. The rate of ATP hydrolysis by MtbRho in presence of polyC was.10-fold less when compared to that of EcRho. Also, as previous studies have shown, MtbRho exhibited a higher Km for ATP than EcRho. The slow rate of ATP hydrolysis indicates the intrinsically low activity of the enzyme. However, when mycobacterial cellular RNA was used, the ATPase rate of MtbRho became similar to previous GDC-0879 observations where E. coli terminator RNAs had been used. Remarkably, the rates of MtbRho and EcRho became comparable in presence of mycobacterial cellular RNA. This indicated that MtbRho could be more proficient in using mycobacterial RNA as substrate than its E. coli homolog. The superior ability of MtbRho to utilize mycobacterial RNA was further evident when, in presence of sdaA RNA, a specific Mtb RNA, MtbRho hydrolysed ATP at a rate that is 2-fold higher than that reported in presence of the E. coli terminators, while EcRho was unable to use the sdaA RNA for ATPase activity.