title: 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.
authors.affiliation: a Department of Biosciences and Bioengineering , Indian Institute of Technology Guwahati , Assam , India.
authors.forename: Himanshu
authors.initials: H
authors.lastname: Sharma
authors.affiliation: a Department of Biosciences and Bioengineering , Indian Institute of Technology Guwahati , Assam , India.
authors.forename: Baskaran
authors.initials: B
authors.lastname: Anand
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D012269:Ribosomal Proteins
keywords: Assembly defect; BiFC; assembly factors; fluorescence complementation; ribosome assembly