In spite of long-standing efforts to prevent and treat tuberculosis, this disease remains a major public health concern. It is estimated that nearly a third of the human population is infected by its causal agent: Mycobacterium tuberculosis (Dye et al., 1999). Adaptation to the various conditions encountered by this pathogen in the establishment of a successful infection is thought to require a strict gene expression program. In prokaryotes, much of this control is obtained at the transcriptional level. Bacterial core RNA polymerase (RNAP) is composed of five subunits (α2ββ′ω) (Gruber & Gross, 2003). An additional subunit, the σ factor (σ), associates with RNAP and provides the promoter recognition function to the holoenzyme complex, thus allowing the expression of a particular group of genes (Gruber & Gross, 2003). Thirteen σ factors are encoded in the genome of M. tuberculosis (Cole et al., 1998). Although some have been characterized, much remains to be determined about their physiological roles and promoter recognition specificities (Manganelli et al., 2004). Furthermore, over 140 putative transcriptional regulators are presumably involved in gene expression modulation in this pathogen (Manganelli et al., 2004).
In vitro transcription assays have been extensively used in the study of transcription regulation in prokaryotes. They provide many mechanistic insights and faithfully mimic many aspects of in vivo transcription. Most importantly, in vitro transcription allows a direct investigation as to the role of RNAP, σ factor(s), promoter DNA or any regulator(s) that may contribute to the expression of a gene of interest. However, these studies may require large amounts of high-quality RNAP that can be difficult to obtain. A major difficulty in RNAP purification lies in complete elimination of contaminating σ factors. Biosafety concerns may also arise if large amounts of pathogenic microorganisms have to be grown in order to proceed with the purification. To circumvent these potential problems, some researchers have used commercially available Escherichia coli RNAP to study transcriptional regulation in other bacteria (Raman et al., 2001; Song et al., 2003; Sun et al., 2004). Such shortcomings could have important consequences as important protein–protein interactions may not be conserved between species (Steffen & Ullmann, 1998; Mencia et al., 1998; Lohrke et al., 1999). Moreover, RNAP from different bacterial species may have distinct properties (Artsimovitch et al., 2000).
Using recombinant proteins to reconstitute a functional enzyme is an interesting alternative to conventional RNAP purification from crude extracts (Tang et al., 1995). The procedure is fast, does not require biosafety containment facilities and yields a σ factor-free preparation. Moreover, hybrid RNAP can easily be generated, and mutations that could otherwise be lethal or could severely impair bacterial growth can be introduced in any of the proteins (Tang et al., 1995; Peck et al., 2002). This work describes the preparation and the use of a recombinant in vitro transcription system for the study of gene expression regulation in M. tuberculosis.