The VapBC toxin-antitoxin (TA) systems were first identified in 2005 and little is known about their contemporary biological function, despite the fact that VapBC TAs are the largest TA family and are widespread in bacteria and archaea (Arcus, Rainey, & Turner, 2005; Gerdes, Christensen, & Lobner-Olsen, 2005). Mycobacterium tuberculosis has a surprisingly large repertoire of 45 VapBC TAs. In contrast Mycobacterium smegmatis, a model organism for M. tuberculosis, contains only one vapBC operon, thereby making it an ideal system to uncover the possible role(s) that VapBC proteins play in mycobacteria. This thesis describes the functional characterisation of VapC from M. smegmatis and two homologues from Pyrobaculum aerophilum, along with biophysical characterisation of the VapBC complex from M. smegmatis. The VapBC proteins from M. smegmatis form a tight complex in a 1:1 ratio and interactions between the proteins result in a tetramer of VapBC heterodimers. VapB is susceptible to proteolytic degradation when not bound to DNA thus hinting at a mechanism for VapC activation. VapC proteins from P. aerophilum (VapCPAE2754 and VapCPAE0151) and VapC from M. smegmatis display Mg²⁺/Mn²⁺ dependent, sequence specific ribonuclease activity. VapC from M. smegmatis targets the AU rich sequences AUAU and AUAA. Whereas VapCPAE2754 and VapCPAE0151 from P. aerophilum both target GGUG and GGGG sequences. These sequences are present in over half the mRNA transcripts encoded in the P. aerophilum genome, making them potent toxins. VapC ribonuclease activity is inhibited when VapC is bound to VapB. When VapC is released from the VapBC complex it cleaves cohorts of mRNA transcripts thereby reducing protein synthesis for this cohort of genes. Microarray analysis revealed that the majority of transcripts downregulated in response to VapC expression in M. smegmatis are involved in carbon utilisation and transport (Robson, 2010). Bioinformatics shows that the target sequence is overrepresented in the downregulated transcripts. The majority of the downregulated genes are in operons, which suggests a mechanism for destabilising mRNA transcripts to regulate specific metabolic processes.