Validation of a Fluorescence-based Screening Concept to Identify Ribosome Assembly Defects in Escherichia Coli

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2014 May 6

PMID 24792169

Citations 9

Authors

Rainer Nikolay

Renate Schloemer

Sabine Schmidt

Silke Mueller

Anja Heubach

Elke Deuerling

Affiliations

Soon will be listed here.

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.

Citing Articles

The proline-rich antimicrobial peptide Api137 disrupts large ribosomal subunit assembly and induces misfolding.

Lauer S, Gasse J, Krizsan A, Reepmeyer M, Sprink T, Nikolay R Nat Commun. 2025; 16(1):567.

PMID: 39794318 PMC: 11723945. DOI: 10.1038/s41467-025-55836-8.

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli.

Matamouros S, Gensch T, Cerff M, Sachs C, Abdollahzadeh I, Hendriks J Nat Commun. 2023; 14(1):5611.

PMID: 37699882 PMC: 10497606. DOI: 10.1038/s41467-023-41176-y.

History-dependent physiological adaptation to lethal genetic modification under antibiotic exposure.

Koganezawa Y, Umetani M, Sato M, Wakamoto Y Elife. 2022; 11.

PMID: 35535492 PMC: 9090333. DOI: 10.7554/eLife.74486.

NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens.

Stukenberg D, Hoff J, Faber A, Becker A Commun Biol. 2022; 5(1):265.

PMID: 35338236 PMC: 8956659. DOI: 10.1038/s42003-022-03150-0.

Ribosome assembly defects subvert initiation Factor3 mediated scrutiny of bona fide start signal.

Sharma H, Anand B Nucleic Acids Res. 2019; 47(21):11368-11386.

PMID: 31586395 PMC: 6868393. DOI: 10.1093/nar/gkz825.

References

Wilson D, Cate J . The structure and function of the eukaryotic ribosome. Cold Spring Harb Perspect Biol. 2012; 4(5). PMC: 3331703. DOI: 10.1101/cshperspect.a011536. View

Tenson T, Lovmar M, Ehrenberg M . The mechanism of action of macrolides, lincosamides and streptogramin B reveals the nascent peptide exit path in the ribosome. J Mol Biol. 2003; 330(5):1005-14. DOI: 10.1016/s0022-2836(03)00662-4. View

Wikstrom P, Bystrom A, Bjork G . Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli. J Mol Biol. 1988; 203(1):141-52. DOI: 10.1016/0022-2836(88)90098-8. View

English B, Hauryliuk V, Sanamrad A, Tankov S, Dekker N, Elf J . Single-molecule investigations of the stringent response machinery in living bacterial cells. Proc Natl Acad Sci U S A. 2011; 108(31):E365-73. PMC: 3150888. DOI: 10.1073/pnas.1102255108. View

Dodd J, Kolb J, Nomura M . Lack of complete cooperativity of ribosome assembly in vitro and its possible relevance to in vivo ribosome assembly and the regulation of ribosomal gene expression. Biochimie. 1991; 73(6):757-67. DOI: 10.1016/0300-9084(91)90055-6. View

Schuwirth B, Borovinskaya M, Hau C, Zhang W, Vila-Sanjurjo A, Holton J . Structures of the bacterial ribosome at 3.5 A resolution. Science. 2005; 310(5749):827-34. DOI: 10.1126/science.1117230. View

Maguire B . Inhibition of bacterial ribosome assembly: a suitable drug target?. Microbiol Mol Biol Rev. 2009; 73(1):22-35. PMC: 2650890. DOI: 10.1128/MMBR.00030-08. View

Traub P, Nomura M . Structure and function of E. coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins. Proc Natl Acad Sci U S A. 1968; 59(3):777-84. PMC: 224743. DOI: 10.1073/pnas.59.3.777. View

Shajani Z, Sykes M, Williamson J . Assembly of bacterial ribosomes. Annu Rev Biochem. 2011; 80:501-26. DOI: 10.1146/annurev-biochem-062608-160432. View

10.

Datsenko K, Wanner B . One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000; 97(12):6640-5. PMC: 18686. DOI: 10.1073/pnas.120163297. View

11.

Bubunenko M, Korepanov A, Court D, Jagannathan I, Dickinson D, Chaudhuri B . 30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15. RNA. 2006; 12(7):1229-39. PMC: 1484426. DOI: 10.1261/rna.2262106. View

12.

Osawa S, Otaka E, Itoh T, Fukui T . Biosynthesis of 50 s ribosomal subunit in Escherichia coli. J Mol Biol. 1969; 40(3):321-51. DOI: 10.1016/0022-2836(69)90158-2. View

13.

Lutz R, Bujard H . Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997; 25(6):1203-10. PMC: 146584. DOI: 10.1093/nar/25.6.1203. View

14.

Bubunenko M, Baker T, Court D . Essentiality of ribosomal and transcription antitermination proteins analyzed by systematic gene replacement in Escherichia coli. J Bacteriol. 2007; 189(7):2844-53. PMC: 1855809. DOI: 10.1128/JB.01713-06. View

15.

Fu Y, Deiorio-Haggar K, Anthony J, Meyer M . Most RNAs regulating ribosomal protein biosynthesis in Escherichia coli are narrowly distributed to Gammaproteobacteria. Nucleic Acids Res. 2013; 41(6):3491-503. PMC: 3616713. DOI: 10.1093/nar/gkt055. View

16.

Nierhaus K, Dohme F . Total reconstitution of functionally active 50S ribosomal subunits from Escherichia coli. Proc Natl Acad Sci U S A. 1974; 71(12):4713-7. PMC: 433966. DOI: 10.1073/pnas.71.12.4713. View

17.

Ramaswamy P, Woodson S . Global stabilization of rRNA structure by ribosomal proteins S4, S17, and S20. J Mol Biol. 2009; 392(3):666-77. PMC: 2763185. DOI: 10.1016/j.jmb.2009.07.032. View

18.

Champney W . Bacterial ribosomal subunit assembly is an antibiotic target. Curr Top Med Chem. 2003; 3(9):929-47. DOI: 10.2174/1568026033452186. View

19.

Kaczanowska M, Ryden-Aulin M . Ribosome biogenesis and the translation process in Escherichia coli. Microbiol Mol Biol Rev. 2007; 71(3):477-94. PMC: 2168646. DOI: 10.1128/MMBR.00013-07. View

20.

Lentini R, Forlin M, Martini L, Del Bianco C, Spencer A, Torino D . Fluorescent proteins and in vitro genetic organization for cell-free synthetic biology. ACS Synth Biol. 2013; 2(9):482-9. DOI: 10.1021/sb400003y. View