Fluorescence-based monitoring of ribosome assembly landscapes

Rainer Nikolay,Renate Schloemer,Silke Mueller,Elke Deuerling

doi:10.1186/s12867-015-0031-y

Rainer Nikolay, Renate Schloemer + Show 2 more

Open Access

https://doi.org/10.1186/s12867-015-0031-y

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Abstract

BackgroundRibosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control.ResultsWe developed a system that allows visualization of intact ribosomal subunits and assembly intermediates (i.e. assembly landscapes) by convenient fluorescence-based analysis. To this end, we labeled the early assembly ribosomal proteins L1 and S15 with the fluorescent proteins mAzami green and mCherry, respectively, using chromosomal gene insertion. The reporter strain harbors fluorescently labeled ribosomal subunits that operate wild type-like, as shown by biochemical and growth assays. Using genetic and chemical perturbations by depleting genes encoding the ribosomal proteins L3 and S17, respectively, or using ribosome-targeting antibiotics, we provoked ribosomal subunit assembly defects. These defects were readily identified by fluorometric analysis after sucrose density centrifugation in unprecedented resolution.ConclusionThis strategy is useful to monitor and characterize subunit specific assembly defects caused by ribosome-targeting drugs that are currently used and to characterize new molecules that affect ribosome assembly and thereby constitute new classes of antibacterial agents.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0031-y) contains supplementary material, which is available to authorized users.

Highlights

Ribosomes and functional complexes of them have been analyzed at the atomic level
Rationale In order to generate a reporter strain suitable for monitoring ribosome assembly landscapes, we selected ribosomal protein candidates from each subunit according to the following criteria [28,29]: The candidates should be i) distant from functional sites, ii) accessible to C-terminal tagging with fluorescent proteins, iii) early assembly proteins [10] and iv) subject of feedback regulation
The described here fluorescence-based assays allow for monitoring of ribosome assembly landscapes in hitherto unmatched resolution

Summary

Introduction

Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control. Reconstitution of intact ribosomal subunits in the test tube is possible using components derived from purified ribosomes, but requires non-physiological conditions, such as high Mg2+ concentration and incubation temperatures of up to 50°C [2,3]. In vivo, this process critically depends on biogenesis factors, which are proteins that process, modify and chaperone rRNA or r-proteins [4,5]. Ribosome assembly is characterized by a highly coordinated sequence of events consisting of rRNA synthesis and r-protein uptake. Since assembly takes place co-transcriptionally (i.e. during rRNA synthesis) there is a hierarchical order of binding events with early-

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