Deletion of Switch 3 Results in an Archaeal RNA Polymerase That Is Defective in Transcript Elongation

Department of Microbiology, Ohio State University, Columbus, OH 43210, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2010; 285(31):23908-15. DOI: 10.1074/jbc.M109.094565
Source: PubMed


Switch 3 is a polypeptide loop conserved in all multisubunit DNA-dependent RNA polymerases (RNAPs) that extends into the main cleft of the RNAP and contacts each base in a nascent transcript as that base is released from the internal DNA-RNA hybrid. Plasmids have been constructed and transformed into Thermococcus kodakaraensis, which direct the constitutive synthesis of the archaeal RNAP subunit RpoB with an N-terminal His(6) tag and the Switch 3 loop either intact (wild-type) or deleted (DeltaS3). RNAPs containing these plasmid-encoded RpoB subunits were purified, and, in vitro, the absence of Switch 3 had no negative effects on transcription initiation or elongation complex stability but reduced the rate of transcript elongation. The defect in elongation occurred at every template position and increased the sensitivity of the archaeal RNAP to intrinsic termination. Comparing these properties and those reported for a bacterial RNAP lacking Switch 3 argues that this loop functions differently in the RNAPs from the two prokaryotic domains. The close structural homology of archaeal and eukaryotic RNAPs would predict that eukaryotic Switch 3 loops likely conform to the archaeal rather than bacterial functional paradigm.

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    • "In T. kodakarensis, a wide range of genes has been disrupted in order to understand their physiological functions such as those involved in transcription and its regulation, DNA replication, and metabolism. The functions of individual transcription factors such as TFB1/2 (Santangelo et al., 2007), RNA polymerase subunits E and F (Hirata et al., 2008), the switch 3 loop of subunit B (Santangelo and Reeve, 2010) have been examined, along with sequences that can promote transcription termination (Santangelo et al., 2009). Deletion of transcription regulator genes, followed by transcriptome analysis, has led to the identification of regulons and the function of these regulators (Kanai et al., 2007, 2010). "
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    ABSTRACT: This section provides an overview of the genetic systems developed in the Archaea. Genetic manipulation is possible in many members of the halophiles, methanogens, Sulfolobus, and Thermococcales. We describe the selection/counterselection principles utilized in each of these groups, which consist of antibiotics and their resistance markers, and auxotrophic host strains and complementary markers. The latter strategy utilizes techniques similar to those developed in yeast. However, Archaea are resistant to many of the antibiotics routinely used for selection in the Bacteria, and a number of strategies specific to the Archaea have been developed. In addition, examples utilizing the genetic systems developed for each group will be briefly described.
    Preview · Article · Oct 2012 · Frontiers in Microbiology
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    • "The switch regions are flexible ‘hinges’ that connect the clamp to the body of the RNAP (Figure 1). In Eσ70, the switch regions have been proposed to ‘sense’ the presence of nucleic acids in the active centre and couple DNA binding with clamp closure by undergoing conformational changes (4,18,19,25–28). The structural rearrangements of all these elements accommodate the requirements of RPo formation. "
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    ABSTRACT: Bacterial RNA polymerases (RNAPs) are targets for antibiotics. Myxopyronin binds to the RNAP switch regions to block structural rearrangements needed for formation of open promoter complexes. Bacterial RNAPs containing the major variant σ54 factor are activated by enhancer-binding proteins (bEBPs) and transcribe genes whose products are needed in pathogenicity and stress responses. We show that (i) enhancer-dependent RNAPs help Escherichia coli to survive in the presence of myxopyronin, (ii) enhancer-dependent RNAPs partially resist inhibition by myxopyronin and (iii) ATP hydrolysis catalysed by bEBPs is obligatory for functional interaction of the RNAP switch regions with the transcription start site. We demonstrate that enhancer-dependent promoters contain two barriers to full DNA opening, allowing tight regulation of transcription initiation. bEBPs engage in a dual switch to (i) allow propagation of nucleated DNA melting from an upstream DNA fork junction and (ii) complete the formation of the transcription bubble and downstream DNA fork junction at the RNA synthesis start site, resulting in switch region-dependent RNAP clamp closure and open promoter complex formation.
    Full-text · Article · Sep 2012 · Nucleic Acids Research
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    • "New avenues, based on advances in T. kodakarensis genetics, permit direct characterization of innumerable archaeal enzymes and their chemistries (Atomi et al., 2001, 2004a; Shiraki et al., 2003; Fukuda et al., 2004, 2008; Rashid et al., 2004; Sato et al., 2004, 2007; Imanaka et al., 2006; Murakami et al., 2006; Orita et al., 2006; Kanai et al., 2007, 2010, 2011; Danno et al., 2008; Fujiwara et al., 2008; Louvel et al., 2009; Yokooji et al., 2009; Borges et al., 2010; Kobori et al., 2010; Morimoto et al., 2010; Matsubara et al., 2011), provide industrially relevant alternative biofuel platforms(Kanai et al., 2005, 2011; Chou et al., 2008; Kim et al., 2010; Atomi et al., 2011; Santangelo et al., 2011; Bae et al., 2012; Davidova et al., 2012), unlock the largely untapped reservoir of archaeal encoded natural products (Atomi, 2005; Kim and Peeples, 2006; Littlechild, 2011; Matsumi et al., 2011; Sato and Atomi, 2011), and offer the opportunity to dissect eukaryotic-like information processing systems composed of minimal components (Yamamoto et al., 2003; Santangelo and Reeve, 2006, 2010a; Kanai et al., 2007; Santangelo et al., 2007, 2008a, 2009; Hirata et al., 2008; Dev et al., 2009; Yamaji et al., 2009; Fujikane et al., 2010; Li et al., 2010, 2011; Ishino et al., 2011; Nunoura et al., 2011; Pan et al., 2011; Santangelo and Artsimovitch, 2011). "
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    ABSTRACT: Thermococcus kodakarensis (T. kodakarensis) has emerged as a premier model system for studies of archaeal biochemistry, genetics, and hyperthermophily. This prominence is derived largely from the natural competence of T. kodakarensis and the comprehensive, rapid, and facile techniques available for manipulation of the T. kodakarensis genome. These genetic capacities are complemented by robust planktonic growth, simple selections, and screens, defined in vitro transcription and translation systems, replicative expression plasmids, in vivo reporter constructs, and an ever-expanding knowledge of the regulatory mechanisms underlying T. kodakarensis metabolism. Here we review the existing techniques for genetic and biochemical manipulation of T. kodakarensis. We also introduce a universal platform to generate the first comprehensive deletion and epitope/affinity tagged archaeal strain libraries.
    Full-text · Article · Jun 2012 · Frontiers in Microbiology
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