Besides ATP, MtbRho hydrolysed GTP while RNA-dependent CTP and UTP hydrolysis was undetectable. Thus, MtbRho can be considered an NTPase, with a substrate preference for ATP and GTP, when provided with its cognate RNAs. The ability to hydrolyze the various NTPs at different levels was mirrored by MtbRho’s ability to bind the various NTPs with varying efficiency. When UVradiation was used to crosslink c-32P-ATP to MtbRho, in presence of unlabeled NTPs, ATP could compete out the crosslinking of c-32P-ATP, but CTP could not. MtbRho is an NTPase that can hydrolyse ATP and GTP efficiently in presence of GC-rich RNA. Although a weaker ATPase compared to the well-characterized EcRho, MtbRho is more efficient in utilizing ATP in presence of its natural substrate i.e. mycobacterial RNAs, compared to EcRho. The intrinsically weak ATPase activity of MtbRho suggests that the enzyme could be a poor translocase as ATP hydrolysis is necessary for powering translocation along the nascent transcript towards RNAP. Notably, it is not the first mycobacterial enzyme shown to have slow catalytic rates. The rates of chain elongation of both M. tuberculosis RNAP and DNA Polymerase is significantly lower than the corresponding E. coli enzymes and the slow rates are considered to be optimized to the slow-growing lifestyle of the bacteria. Thus, the ATPase rate of MtbRho could be an optimized rate evolved so that termination is functionally in sync with the slowly transcribing MtbRNAP. A stronger MtbRho ATPase, in contrast, could possibly result in swift and premature termination, which would be catastrophic for gene expression. The ability of MtbRho to hydrolyse ATP in presence of polyA and polyU, RNA substrates that are inaccessible to EcRho, shows that such broad-RNA specificity is a conserved feature of actinobacterial Rho. Such broad specificity for RNAs could be useful in interacting with a larger number of RNA molecules, both for normal, intergenic rho-dependent termination as well as silencing of xenogenic DNA. It may be indicative of the greater importance of Rho-assisted termination in Mtb and other actinobacteria, which have fewer intrinsic terminators. The inefficient use of CTP by MtbRho also parallels earlier studies with Rho homologs from M. luteus and Streptomyces lividans. The GANT61 cost latter two enzymes also hydrolyzed CTP very inefficiently. This strategy could be possibly an adaptation to spare CTP for transcription of GC-rich transcriptomes of these actinobacteria. The conserved amino acid composition of the N-terminal additional region is indicative of its functional importance and it has been hypothesized to have a role in binding to GC-rich RNA ; M. luteus Rho has been shown to terminate transcription by E. coli RNAP at sites where EcRho cannot terminate, and this is considered indicative that the ‘larger’ Rho are more efficient for terminating on GC-rich RNAs.