Analysis of Bacterial RNA Polymerase

Our experts have a keen interest in the study of liquid-liquid and liquid-solid phase transitions in bacterial cells. We have cutting-edge super-resolution imaging or infinite diffraction microscopy apparently combined with single molecule trafficking methods, and computer analysis platforms to analyze key microbial biomolecular condensates undergoing LLPS, as well as the formation and organization of biomolecular condensates within the intracellular space. Here, CD BioSciences is committed to analyzing intrinsically disordered bacterial RNA polymerase (RNAP).

Introduction of Bacterial RNA Polymerase

RNAP is the enzyme responsible for RNA transcription. They initiate the process at a specific DNA promoter sequence, and their activities are regulated by a number of transcription factors. In contrast to eukaryotes, which contain three different types of enzymes, bacteria have the simplest form of RNAP to synthesize different kinds of RNA. Unlike other DNA-binding proteins, RNAP interacts extensively and dynamically (specifically and non-specifically) with DNA and maintains them at a certain distance. These interactions are controlled by DNA sequence, DNA topology, and many regulatory factors. Since they exhibit some signature features, studies suggest that RNAP are biomolecular condensates assembled by liquid-liquid phase separation (LLPS).

Fig. 1. Structural overview of the RNAP core. (Lee J, et al., 2016)

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The structure of RNAP is essential to fully interpret the vast amount of biochemical, biophysical, and genetic data on RNAP. Our laboratory has electron microscopy to perform comprehensive structural studies of the stable structural domains and subcomplexes within RNAP from E. coli, providing you with information on the X-ray crystal structure of bacterial RNAP.

Based on key structural and biochemical information of RNAP, our technical team is dedicated to analyzing RNAP-DNA/protein interactions during bacterial initiation. CD BioSciences provides professional services to analyze the structural dynamics and biochemical functions of the intrinsically disordered RNAP in bacteria. We provide DNA binding or transcription factory models to explain RNAP clustering. In addition, we can combine traditional chemical and genetic perturbations with single-molecule tracking to study bacterial RNAP clustering.

• Characterization of RNAP LLPS in Vitro
  To test the phase separation ability of bacterial RNAP, we monitored their behavior in vitro by purifying GFP-RNAP and using complementary biochemical assays. The specific procedure was as follows:
  (1) Cells were treated with the fatty alcohol 1,6-hexanediol to induce LLPS.
  (2) Search for condensed protein droplets by fluorescence microscopy.
  (3) Measure the protein concentration of RNAP solution after centrifugation.
  (4) Modulate the affinity of intermolecular interactions by varying the salt concentration in the buffer and plot the phase diagram of RNAP.
• Characterization of RNAP LLPS in Vivo
  NusA is an anti-termination factor that interacts with RNAP. We use the strategy of the lacO/LacI system to analyze the interaction of NusA with RNAP. We can generate two constructs (LacI-mNeonGreen alone and LacI-NusA-mNeonGreen) and express them in cells to characterize the condensates. We also provide single molecule tracking to analyze the dynamics of RNAP fractions under different growth conditions.
• Development of Bacterial RNAP-Inhibitor Complexes
  RNAP is essential for bacterial growth and survival and has characteristics that distinguish it from its mammalian counterpart. Our experts are committed to developing RNAP as an important target for antimicrobial chemotherapy and exploring RNAP-assembled inhibitors.

CD BioSciences offers a simple modular biomolecular platform to characterize the LLPS of intrinsically disordered RNAP. We aim to analyze the molecular components and interactions that drive RNAP assembly in bacteria and determine their effects on gene expression, ribosome biogenesis, and cell growth and size. If you have any special requirements for our services, please feel free to contact us.