Fluorescence bimolecular complementation enables facile detection of ribosome assembly defects in Escherichia coli

Abstract

Assembly factors promote the otherwise non-spontaneous maturation of ribosome under physiological conditions inside the cell. Systematic identification and characterization of candidate assembly factors are fraught with bottlenecks due to lack of facile assay system to capture assembly defects. Here, we show that bimolecular fluorescence complementation (BiFC) allows detection of assembly defects that are induced by the loss of assembly factors. The fusion of N and C-terminal fragments of Venus fluorescent protein to the ribosomal proteins uS13 and uL5, respectively, in Escherichia coli facilitated the incorporation of the tagged uS13 and uL5 onto the respective ribosomal subunits. When the ribosomal subunits associated to form the 70S particle, the complementary fragments of Venus were brought into proximity and rendered the Venus fluorescent. Assembly defects that inhibit the subunits association were provoked by either the loss of the known assembly factors such as RsgA and SrmB or the presence of small molecule inhibitors of ribosome maturation such as Lamotrigine and several ribosome-targeting antibiotics and these showed abrogation of the fluorescence complementation. This suggests that BiFC can be employed as a surrogate measure to detect ribosome assembly defects proficiently by circumventing the otherwise cumbersome procedures. BiFC thus offers a facile platform not only for systematic screening to validate potential assembly factors but also to discover novel small molecule inhibitors of ribosome assembly toward mapping the complex assembly landscape of ribosome.

Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function

“…To verify this newly identified SOD2 domain in hsp70-mediated binding, we used bimolecular fluorescence complementation (BiFC) analysis to directly visualize SOD2/hsp70 interactions in live cells (22)(23)(24). Hsp70 constructs were made with deletion of amino acid residues 393-537 to express recombinant hsp70 mutant (hsp70⌬393-537) protein that could not FIGURE 1.…”

Role of Hibernation Promoting Factor in Ribosomal Protein Stability during Pseudomonas aeruginosa Dormancy

“…The supernatant was removed and the cell pellets were frozen at −80 °C. The cells were resuspended in 50 µL ice-cold Buffer A [ 53 ], brought to 1 mL with a final concentration of 0.7 mM lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5× CelLytic TM B (Sigma-aldrich, Burlington, MA, USA), 3 mM Magnesium, and diethyl pyrocarbonate (DEPC) treated water. Cells were lysed by eight freeze-thaw cycles consisting of 10 min at −80 °C followed by 2 min in a 37 °C water bath.…”

“…The cell lysates were spun at 20,000× g for 45 min at 4 °C to remove cellular debris. The supernatants were collected and ribosomes were separated using Buffers A, B, and C from [ 53 ] according to the previously published crude preparation protocol in [ 54 ]. Briefly, the supernatants were adjusted to a final volume of 6 mL with Buffer A and slowly added to RNase-free ultracentrifuge tubes (Beckman Coulter, Indianapolis, IN, USA) containing 6 mL Buffer B.…”

Identification of first active compounds in drug discovery. how to proceed?

“…Specific target-based screens like protein-protein interactions can also be conducted using cell-based assays. For example, bimolecular complementation assays, also known as PCA (Protein-fragments Complementation assays) have been developed in the last decade (Kodama and Hu, 2012;Sharma and Anand, 2016;Bellón-Echeverría et al, 2018). In these assays, a fluorescent reporter protein is divided into two non-functional fragments.…”