Validation of a fluorescence-based screening concept to identify ribosome assembly defects inEscherichia coli

Abstract

While the structure of mature ribosomes is analyzed in atomic detail considerably less is known about their assembly process in living cells. This is mainly due to technical and conceptual hurdles. To analyze ribosome assembly in vivo, we designed and engineered an Escherichiacoli strain—using chromosomal gene knock-in techniques—that harbors large and small ribosomal subunits labeled with the fluorescent proteins EGFP and mCherry, respectively. A thorough characterization of this reporter strain revealed that its growth properties and translation apparatus were wild-type like. Alterations in the ratio of EGFP over mCherry fluorescence are supposed to indicate ribosome assembly defects. To provide proof of principle, subunit specific assembly defects were provoked and could be identified by both manual and fully automated fluorometric in vivo assays. This is to our knowledge the first methodology that directly detects ribosome assembly defects in vivo in a high-throughput compatible format. Screening of knock-out collections and small molecule libraries will allow identification of new ribosome assembly factors and possible inhibitors.

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

“…This necessitated that the ratio of the intensity of both colors is characterized which is further subjected to variability under different experimental conditions. 31,36 In our design, this is circumvented by adopting BiFC as a surrogate measure to monitor the state of the ribosome assembly. Since the fluorophore maturation in Venus -owing to the requirement of spatial proximity of the complementary parts -takes place on the fully assembled ribosomes, BiFC analysis based on purified crude ribosomes alleviates false positives to a large extent without compromising the sensitivity.…”

“…This leads to the cascading effect of diminishing the production of r-proteins too whose dosage insufficiency becomes rate limiting thus provoking the ribosome assembly defects indirectly. 30,31 On the contrary, Ampicillin, which is an inhibitor of transpeptidases prevents cell wall synthesis and therefore anticipated to not induce any ribosome assembly defects. We assessed BiFC from crude ribosomes purified from antibiotics treated SLV NC relative to that of untreated SLV NC .…”

Fluorescent polysome profiling reveals stress-mediated regulation of HSPA14-ribosome interactions

“…This method is laborious, especially when looking to compare different conditions. More recently, fluorescence monitoring of polysome fractions was performed after fractionation, using fluorescently tagged ribosomal proteins in E.coli, to examine ribosome assembly intermediates (Nikolay, Schloemer et al, 2014), improving the efficiency of the traditional method.…”

Assembly Defects Abrogate Proofreading by Initiation Factors and License the Entry of Premature Ribosomes into the Translation Cycle:

“…The 70S-IC eventually enters the elongation phase of the translation and drives protein synthesis. Disruptions in translation are known to hinder the biogenesis of ribosomes by skewing ration of r-Proteins to r-RNA (Nikolay et al, 2014; Siibak et al, 2009). Additionally, the suboptimal maturation of ribosomes is implicated in several disorders collectively referred to as ribosomopathies (Danilova and Gazda, 2015; Freed et al, 2010; Mills and Green, 2017).…”