Abstract
Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed.
Highlights
The ability to respond to changes in nutritional environment is a universal need inherent in all cells and is characterized by rapid global changes in gene expression
Study of the nutritional response in Escherichia coli has detailed a novel mechanism of modulating transcription initiation, both positively and negatively, through the use of a single small nucleotide effector, guanosine tetraphosphate, that interacts with RNA polymerase [1]
Loss of DksA function causes permanent downregulation of several amino acid biosynthetic and transport pathways and results in an inability of E. coli cells to grow on minimal media without supplementation of required amino acids [4,6,11]
Summary
The ability to respond to changes in nutritional environment is a universal need inherent in all cells and is characterized by rapid global changes in gene expression. In E. coli, the accumulation of ppGpp causes rapid effects on transcription; ppGpp binds to RNA polymerase, provoking an alteration in transcription kinetics that is proposed to result from a reduction in open complex stability [2] Such effects include, but are not limited to, upregulation of amino acid biosynthesis and transport genes, as well as genes involved in stasis/stress survival, and downregulation of translational components such as rRNA and tRNA genes [3]. At the tip of the coiled coil region, two invariant aspartic acid residues, Asp and Asp, are thought to coordinate the ppGpp bound Mg+2 ion to effectively position ppGpp near the active site, and allow it to exert its transcriptional modulation effects [15].
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