Transcriptional control of bacterial sporulation
Prof. Markus Wahl
Bacterial sporulation is a developmental program that results in formation of highly resistant spores. The execution of this program depends on concerted changes in gene expression that are mediated by RNA polymerase (RNAP) and a cascade of alternative σ factors. Preliminary results revealed surprising effects of non-essential subunits of RNAP, δ and ω, on the efficiency of sporulation in Bacillus subtilis. Consequently, we propose to systematically explore the structure and function of transcription complexes and the dynamics of their composition during sporulation in Bacillus subtilis, focusing on the interplay between RNAP, δ, ω, σ factors and other associating proteins. The project will be conducted in collaboration with the group of Libor Krásný, Academy of Sciences of the Czech Republic, who will investigate changes in cell morphology, gene expression and the RNAP protein interactome during sporulation. Our group will contribute structural and structure-based functional investigations on how δ and ω modulate σ factor switching during sporulation. The project will establish a basis for our understanding of the mechanistic aspects of the transcriptional machinery during sporulation, a process extensively utilized in food, feed and pharmaceutical applications.
Presently, no structure of a holoenzyme or initiation complex based on a δ/ω-containing RNAP is available. RNAP in the presence of δ and σ factors can bind promoters and form closed complexes. However, based on our structural and functional analyses of δ in the context of RNAP-HelD recycling complexes, we suggest that holoenzymes in the presence of δ are inhibited with respect to establishing contacts with the downstream DNA, due to the tendency of the δ C-terminal region (CTR) to occupy the main channel. RNAP-downstream DNA contacts are required for the transition to an open complex. We hypothesize that δ hinders open complex formation, and thus transcription initiation, with different efficiencies for different σ factors. ω, in turn, may further modulate the δ/σ interplay by virtue of its own binding competition with δ. To test these hypotheses, we will conduct cryogenic electron microscopy (cryoEM)-based structural analyses on B. subtilis RNAP holoenzymes and then progress to closed complexes, open complexes and initial transcription complexes. We will investigate complexes based on σH, σE and σF, and will compare complexes containing or lacking δ and/or ω. In addition, we will include further interacting proteins identified by our collaborators.
We have established efficient strategies to purify active B. subtilis RNAP (full core enzyme or core enzyme lacking δ, ω or both) from strains that harbor a His-tagged β‘ subunit or from plasmids. Likewise, protocols for the recombinant production and purification of δ, δ N-terminal domain, ω, σH, σE and σF have been established. Other proteins of interest will be produced recombinantly in E. coli or B. subtilis and purified by similar strategies, making use of cleavable affinity tags. DNAs harboring specific promoter sequences without/with artificial transcription bubbles and short RNAs (below ca. 30 nts; if required for elongation complexes) will be chemically synthesized (commercial sources). Longer RNAs, if required, will be produced by in vitro transcription with T7 RNA polymerase. To assess the suitability of the samples for structural studies, we will test the fold stability, mono-dispersity and interaction potential of purified proteins, using analytical gel filtration chromatography, EMSA, pull-down assays, HPLC/multi-angle light scattering, isothermal titration calorimetry, Biacore analysis, microscale thermophoresis, fluorescence anisotropy, CD spectroscopy, CD-based thermal melting or ThermoFluor analysis. These techniques are routinely used in our lab.
Reconstitution of complexes and cryoEM analyses will be guided by our established strategies. Briefly, complexes will be reconstituted from separately produced, recombinant proteins, synthetic DNAs and, if applicable, RNAs. Components will be mixed in defined order with intermittent incubation, and stable complexes will be purified by gel filtration or glycerol gradient centrifugation. Closed and open complexes will be obtained from holoenzymes by adding σ factor-specific promoter DNA excluding (closed) or including (open) an unpaired region. Open complexes may also be accessible with paired DNA by incubation at elevated temperatures. Initial transcription complexes will be obtained from open complexes by adding all but one NTP to allow transcription of a short RNA product. Functionality of the assembled complexes will be probed by testing their ability to bind promoter DNA (holoenzymes), synthesize short transcripts (open complexes) or mediate single nucleotide addition or run-off transcription (elongation complexes). Reconstituted, active complexes will be optimized for single-particle cryoEM-based structural analysis using well-established routines in our lab. We will initially assess and optimize the quality of the samples by negative stain EM on an in-house Talos L120C TEM. Grid preparation for cryoEM will involve systematic testing of different hole diameters, grid surfaces, buffer conditions (e.g., detergents) and sample concentrations. Checking of particle integrity and initial cryoEM data collection will be carried out on an in-house 200 kV Talos Arctica cryoTEM equipped with a direct electron detector (DED). Sufficient data will be collected to ensure random particle orientations under the chosen conditions. Large datasets for high-resolution structure analyses will be collected on an in-house 300 kV Krios Titan cryoTEM with DED. Structures will be solved by established procedures, including hierarchical compositional/conformational sorting. If complexes disintegrate during purification or grid preparation, we will stabilize them by mild crosslinking in solution or by employing the GraFix technique. Facile access to the required high-end TEMs is provided by the core facility BioSupraMol of Freie Universität Berlin (https://www.biosupramol.de/en). Cross-linking/mass spectrometry (CLMS; supported by the BioMS unit of the BioSupraMol core facility) will be conducted to validate cryoEM structures and to elucidate local structural features in more poorly resolved map regions, as we have done in the past. Guided by the atomic structures, we will alter interacting residues/regions in δ, ω or the σ factors and study the effects of such alterations on holoenzyme or initiation complex assembly as well as on transcription initiation in vitro, using techniques outlined above.
Overall, this project will provide a comprehensive model of the functioning of the transcription machinery during sporulation. Sporulation-related genes affected by δ and/or ω will be identified and roles of δ and/or ω on respective promoters will be elucidated. The sporulation-specific, dynamic interactome of RNAP and its dependence on δ and ω will be revealed. New, sporulation-related binding partners of RNAP will be identified and validated, extending our knowledge of stage-specific transcription factors. The structures of selected sporulation-specific RNAP holoenzymes and initiation complexes will be determined, allowing us to propose mechanistic, testable models of the functioning of the transcription machinery during sporulation. Thus, the project results will provide a comprehensive view of the complex sporulation process from the transcription perspective.