The transcription machinery in the phylum spirochetes is poorly characterized at the molecular level yet is of significant evolutionary and medical importance. Pathogenic spirochetes can 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 non-pathogenic spirochete species exist. Spirochetal RNA polymerases (RNAPs) are naturally resistant to rifampicin (Rif), the best-known transcription inhibitor in clinical use. Spirochetes evolved independently from other bacterial phyla and are not closely related to the well-established model organisms Escherichia coli (Eco) and Bacillus subtilis (Bsu), suggesting that the regulation of transcription in spirochetes includes distinct and novel strategies. Transcription is the first step in the highly regulated process of gene expression. It 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 a Sigma factor (holoenzyme), recognizes promoter motifs on the DNA template and starts RNA synthesis. Many regulatory factors are associated with RNAP during initiation and modulate its activity, including CarD, GreA and DksA. Sequence alignments identified additional or distinct domains of some of these transcription factors in spirochetes compared to most other bacteria phyla, suggesting that they may act through different molecular mechanisms. To investigate transcription mechanisms regulated by CarD, GreA, and DksA, we reconstituted initiation complexes with each initiation factor and determined their structures using cryo-electron microscopy (cryo-EM). Here, I present the cryo-EM structures of Spirochaeta africana (Sfc) RNAP open promoter complex (RPo) in the presence or absence of CarD. Sfc RNAP, together with Sfc σ 70 , binds to the promoter DNA in an open conformation in which the duplex DNA is unwounded and the transcription bubble is formed. The structures suggest that Sfc RPo is unstable and could be stabilized by Sfc CarD via binding to the upstream template DNA (tDNA) of the transcription bubble. I also present the cryo-EM structure of the Sfc RPo in complex with GreA, indicating that Sfc GreA, like Sfc CarD, can stabilize the open promoter complex. This stabilization is achieved through the CarD-like domain, confirming the role of Sfc GreA as an initiation factor. During transcription elongation, transcription factors NusG and NusA are recruited by RNAP in most operons, while LoaP comes specifically in certain operons, promoting the RNA chain extended correctly. In addition, Sfc NusA has two additional acidic disordered loops on the AR1 and AR2 domains compared to Eco NusA. To investigate the functions of Sfc NusA and LoaP, we reconstituted elongation complexes with NusG/NusA and LoaP/NusA, and determined their structures using cryo-EM. I present the cryo-EM structures of Sfc RNAP in complex with NusG/NusA and LoaP/NusA, respectively. Although Sfc NusA does not change much the conformation of RNAP in the presence of NusG, it cooperates with LoaP to push nucleic acids away from RNAP, ensuring that the correct transcription elongation factor is recruited into the appropriate operon. The structures provide essential insights into the overall architecture of Sfc RNAP, the initiation and elongation complexes, and form the basis for further functional and structural analyses of spirochete-specific transcription factors and their regulation.
