Structural basis of promoter melting in Spirochaeta africana
Tingting Wang1, Tarek Hilal2, Janne Mäkinen3, Sari Paavilainen3, Bernhard Loll1, Nelly Said1, Georgiy A. Belogurov3, Markus C. Wahl1,4*
1 Laboratory of Structural Biochemistry, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
2 Research Center of Electron Microscopy and Core Facility BioSupraMol, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
3 Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland.
4 Macromolecular Crystallography, Helmholtz-Zentrum Berlin für Materialien und Energie, Berlin, Germany.
*Corresponding author: Markus C. Wahl
*E-mail: markus.wahl@fu-berlin.de
The transcription systems in the phylum spirochaetes are poorly characterized at the molecular level yet are of significant evolutionary and medical importance. Pathogenic spirochaetes easily move through the mammalian tissues, penetrate blood vessels, cross the blood-brain barrier and cause serious diseases, such as lyme disease, relapsing fever and syphilis. At the same time, many spirochaetes are non-pathogenic free-living species. Notably, spirochetal RNA polymerases (RNAPs) are naturally resistant to rifampicin, the best-known transcription inhibitor in clinical use[1]. Spirochaetes evolved independently from other bacterial phyla and are not related to the well-established model organisms Escherichia coli and Bacillus subtilis, suggesting that the regulation of transcription include distinct and novel strategies.
Transcription is the first event in the highly regulated process of gene expression and is divided into three phases, initiation, elongation and termination that determines the start and the end of the transcription unit. To initiate transcription, RNAP together with sigma factor (holoenzyme) recognizes promoter motifs on the DNA template and start RNA synthesis. Many regulatory factors associate with RNAP during the initiation step and modulate its activity, among them DksA[2], CarD[3] and GreA/B[4]. Sequence alignments identified additional or distinct domains of some of these transcription factors in spirochaetes compared to most other bacteria phyla suggesting they might act through a different molecular mechanism. Here we present the cryogenic electron microscopy (cryoEM) structures of Spirochaeta africana (Sfc) RNAP in complex with sigma factor 70 (σ70) and promoter DNA at 3.0–3.4 Å resolution. SfcRNAP together with the housekeeping Sfcσ70 factor bind to the promoter DNA in an open complex in which the duplex DNA is unwounded and the transcription bubble is formed. The structure reveals important insights into the overall architecture of SfcRNAP and the initiation complex and forms the basis for further functional and structural analyses of spirochaete-specific transcription factors and their regulation.
Figure 1. Sfcσ70 initiation complex. A. Promoter DNA scaffold (-46 to +20) used for cryoEM. The -35 and -10 motifs are indicated. +1 refers to the transcriptional start site. B. Overall architecture of the SfcRNAP-σ70 open promoter complex. Subunits are indicated.
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