The functional importance of the additional N-terminal fragment in MtbRho is further evident from the inability of the truncated protein, which lacks the subdomain, to complement EcRho. It is notable that while N-229 can SJN 2511 stably interact with mycobacterial RNA, it failed to bind efficiently to poly-dC80. In contrast, full-length MtbRho can interact with both the substrates. The primary RNA-binding motifs of Rho are known to that have a preference for C-rich, unstructured RNA and their absence in N-229 could have compromised its ability to interact with poly-dC80. It is also possible that the extended N-terminal subdomain has evolved to bind efficiently to longer RNA molecules, such as the sdaA RNA in this study. Thus, it seems that MtbRho, while retaining ability to bind to the canonical, C-rich substrates of Rho, has a gain-of-function whereby the extended N-terminal region can facilitate binding to mycobacterial RNA. Since MtbRho is a weaker ATPase, interacting with long RNA could result in efficient spooling of RNA around itself and facilitate catching up with RNAP to bring about termination in a largely ATP-independent manner. The inability of MtbRho to complement the strain E.coli AMO14 could be attributed to several reasons. The N-terminal subdomain of MtbRho can facilitate binding to RNA that is not a natural substrate for EcRho. This, in turn, could lead to spurious unregulated termination by MtbRho in the E. coli strain. Alternatively, the lower ATPase activity of MtbRho could result in inefficient termination, especially since rate of transcription elongation by E.coli RNAP is considerably faster than that of mycobacterial RNAP. Approximately 50% of E. coli genes rely on Rho for termination. Hence, delayed or imprecise termination could lead to run-off transcripts, ectopic expression from genomic islands and cryptic prophages. The formation of a distinct hexameric species by glutaraldehyde crosslinking indicates that MtbRho can exist as a hexamer even in absence of its substrate RNA and ATP. DLS and analytical gel filtration studies confirm that at least a fraction of MtbRho exists as hexamer in solution. This hexameric population could provide a ‘readily available’ termination-proficient MtbRho within Mtb cells. This is in contrast to EcRho which forms hexamer only under catalytic conditions. Our results show that the oligomerization status of MtbRho is significantly influenced by its interaction with RNA. Although a fraction of MtbRho always exists as a,400 kDa hexamer, it becomes the predominant form in presence of RNA. The availability of intracellular RNA would thus, act as a cue and ‘activate’ MtbRho to its functional hexameric form. This, in turn, could achieve a functional coordination between MtbRNAP and MtbRho to ensure an orchestrated transcription elongation and termination. In this context, it is noteworthy that the hexamers formed by two Mtb elongation/termination factors.