Fluorescence-based monitoring of ribosome assembly landscapes

BMC Molecular Biology

Elke Deuerling¹  Silke Mueller³  Renate Schloemer¹  Rainer Nikolay² 

[1] Molecular Microbiology, University of Konstanz, Constance 78457, Germany;Current address: Institute of Medical Physics and Biophysics, Charité-Universitaetsmedizin Berlin, Berlin 10117, Germany;Screening Center Konstanz, University of Konstanz, Constance 78457, Germany

关键词: High throughput screening;    λ-red recombineering;    Knock out;    Antimicrobials;    Fluorescent proteins;    Ribosome biogenesis;    Ribosome assembly;   

Others  :  1134567
DOI  :  10.1186/s12867-015-0031-y

 received in 2014-09-12, accepted in 2015-02-03,  发布年份 2015

【 摘 要 】

Background

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.

Results

We 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.

Conclusion

This 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.

【 授权许可】

   
2015 Nikolay et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]de Narvaez CC, Schaup HW: In vivo transcriptionally coupled assembly of Escherichia coli ribosomal subunits. J Mol Biol 1979, 134(1):1-22.
• [2]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.
• [3]Nierhaus KH, 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.
• [4]Kaczanowska M, Ryden-Aulin M: Ribosome biogenesis and the translation process in Escherichia coli. Microbiol Mol Biol Rev 2007, 71(3):477-94.
• [5]Shajani Z, Sykes MT, Williamson JR: Assembly of bacterial ribosomes. Annu Rev Biochem 2011, 80:501-26.
• [6]Nierhaus KH: The assembly of prokaryotic ribosomes. Biochimie 1991, 73(6):739-55.
• [7]Nomura M, Gourse R, Baughman G: Regulation of the synthesis of ribosomes and ribosomal components. Annu Rev Biochem 1984, 53:75-117.
• [8]Marvaldi J, Pichon J, Delaage M, Marchis-Mouren G: Individual ribosomal protein pool size and turnover rate in Escherichia coli. J Mol Biol 1974, 84(1):83-96.
• [9]Ulbrich B, Nierhaus KH: Pools of ribosomal proteins in Escherichia coli. Studies on the exchange of proteins between pools and ribosomes. Eur J Biochem 1975, 57(1):49-54.
• [10]Chen SS, Williamson JR: Characterization of the ribosome biogenesis landscape in E. coli using quantitative mass spectrometry. J Mol Biol 2013, 425(4):767-79.
• [11]Lindahl L: Intermediates and time kinetics of the in vivo assembly of Escherichia coli ribosomes. J Mol Biol 1975, 92(1):15-37.
• [12]Maguire BA: Inhibition of bacterial ribosome assembly: a suitable drug target? Microbiol Mol Biol Rev 2009, 73(1):22-35.
• [13]Basturea GN, Zundel MA, Deutscher MP: Degradation of ribosomal RNA during starvation: comparison to quality control during steady-state growth and a role for RNase PH. RNA 2011, 17(2):338-45.
• [14]Deutscher MP: Maturation and degradation of ribosomal RNA in bacteria. Prog Mol Biol Transl Sci 2009, 85:369-91.
• [15]Champney WS: The other target for ribosomal antibiotics: inhibition of bacterial ribosomal subunit formation. Infect Disord Drug Targets 2006, 6(4):377-90.
• [16]Siibak T, Peil L, Xiong L, Mankin A, Remme J, Tenson T: Erythromycin- and chloramphenicol-induced ribosomal assembly defects are secondary effects of protein synthesis inhibition. Antimicrob Agents Chemother 2009, 53(2):563-71.
• [17]Siibak T, Peil L, Donhofer A, Tats A, Remm M, Wilson DN, et al.: Antibiotic-induced ribosomal assembly defects result from changes in the synthesis of ribosomal proteins. Mol Microbiol 2011, 80(1):54-67.
• [18]Sykes MT, Shajani Z, Sperling E, Beck AH, Williamson JR: Quantitative proteomic analysis of ribosome assembly and turnover in vivo. J Mol Biol 2010, 403(3):331-45.
• [19]Talkington MW, Siuzdak G, Williamson JR: An assembly landscape for the 30S ribosomal subunit. Nature 2005, 438(7068):628-32.
• [20]Popova AM, Williamson JR: Quantitative analysis of rRNA modifications using stable isotope labeling and mass spectrometry. J Am Chem Soc 2014, 136(5):2058-69.
• [21]Siibak T, Remme J: Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified. RNA 2010, 16(10):2023-32.
• [22]Guo Q, Goto S, Chen Y, Feng B, Xu Y, Muto A, et al.: Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process. Nucleic Acids Res 2013, 41(4):2609-20.
• [23]Li N, Chen Y, Guo Q, Zhang Y, Yuan Y, Ma C, et al.: Cryo-EM structures of the late-stage assembly intermediates of the bacterial 50S ribosomal subunit. Nucleic Acids Res 2013, 41(14):7073-83.
• [24]Clatterbuck Soper SF, Dator RP, Limbach PA, Woodson SA: In vivo X-ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates. Mol Cell 2013, 52(4):506-16.
• [25]Jomaa A, Jain N, Davis JH, Williamson JR, Britton RA, Ortega J: Functional domains of the 50S subunit mature late in the assembly process. Nucleic Acids Res 2014, 42(5):3419-35.
• [26]Nikolay R, Schloemer R, Schmidt S, Mueller S, Heubach A, Deuerling E: Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli. Nucleic Acids Res 2014, 42(12):e100.
• [27]Karasawa S, Araki T, Yamamoto-Hino M, Miyawaki A: A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling. J Biol Chem 2003, 278(36):34167-71.
• [28]Schuwirth BS, Borovinskaya MA, Hau CW, Zhang W, Vila-Sanjurjo A, Holton JM, et al.: Structures of the bacterial ribosome at 3.5 A resolution. Science 2005, 310(5749):827-34.
• [29]Zhou J, Lancaster L, Donohue JP, Noller HF: Crystal structures of EF-G-ribosome complexes trapped in intermediate states of translocation. Science 2013, 340(6140):12360861-9.
• [30]Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al.: Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006, 2:1-11.
• [31]Philippe C, Eyermann F, Benard L, Portier C, Ehresmann B, Ehresmann C: Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site. Proc Natl Acad Sci U S A 1993, 90(10):4394-8.
• [32]Subramanian AR, Dabbs ER: Functional studies on ribosomes lacking protein L1 from mutant Escherichia coli. Eur J Biochem 1980, 112(2):425-30.
• [33]Portier C, Dondon L, Grunberg-Manago M: Translational autocontrol of the Escherichia coli ribosomal protein S15. J Mol Biol 1990, 211(2):407-14.
• [34]Yates JL, Nomura M: Feedback regulation of ribosomal protein synthesis in E. coli: localization of the mRNA target sites for repressor action of ribosomal protein L1. Cell 1981, 24(1):243-9.
• [35]Datsenko KA, Wanner BL: 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.
• [36]Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL: An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 2000, 97(11):5978-83.
• [37]Lhoest J, Colson C: Cold-sensitive ribosome assembly in an Escherichia coli mutant lacking a single methyl group in ribosomal protein L3. Eur J Biochem 1981, 121(1):33-7.
• [38]Herzog A, Yaguchi M, Cabezon T, Corchuelo MC, Petre J, Bollen A: A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli. Mol Gen Genet 1979, 171(1):15-22.
• [39]Maguire BA, Wild DG: The roles of proteins L28 and L33 in the assembly and function of Escherichia coli ribosomes in vivo. Mol Microbiol 1997, 23(2):237-45.
• [40]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.
• [41]Dodd J, Kolb JM, 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.
• [42]Chittum HS, Champney WS: Erythromycin inhibits the assembly of the large ribosomal subunit in growing Escherichia coli cells. Curr Microbiol 1995, 30(5):273-9.
• [43]Usary J, Champney WS: Erythromycin inhibition of 50S ribosomal subunit formation in Escherichia coli cells. Mol Microbiol 2001, 40(4):951-62.
• [44]Mehta R, Champney WS: 30S ribosomal subunit assembly is a target for inhibition by aminoglycosides in Escherichia coli. Antimicrob Agents Chemother 2002, 46(5):1546-9.
• [45]Mehta R, Champney WS: Neomycin and paromomycin inhibit 30S ribosomal subunit assembly in Staphylococcus aureus. Curr Microbiol 2003, 47(3):237-43.
• [46]Ulbrich B, Mertens G, Nierhaus KH: Cooperative binding of 3’-fragments of transfer ribonucleic acid to the peptidyltransferase center of Escherichia coli ribosomes. Arch Biochem Biophys 1978, 190(1):149-54.
• [47]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.
• [48]Wilson DN: Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat Rev Microbiol 2014, 12(1):35-48.
• [49]Borovinskaya MA, Pai RD, Zhang W, Schuwirth BS, Holton JM, Hirokawa G, et al.: Structural basis for aminoglycoside inhibition of bacterial ribosome recycling. Nat Struct Mol Biol 2007, 14(8):727-32.
• [50]Wang L, Pulk A, Wasserman MR, Feldman MB, Altman RB, Cate JH, et al.: Allosteric control of the ribosome by small-molecule antibiotics. Nat Struct Mol Biol 2012, 19(9):957-63.
• [51]Hosokawa K, Nomura M: Incomplete ribosomes produced in chloramphenicol- and Puromycin-inhibited Escherichia Coli. J Mol Biol 1965, 12:225-41.
• [52]Cortay JC, Cozzone AJ: Effects of aminoglycoside antibiotics on the coupling of protein and RNA syntheses in Escherichia coli. Biochem Biophys Res Commun 1983, 112(3):801-8.
• [53]Mulder AM, Yoshioka C, Beck AH, Bunner AE, Milligan RA, Potter CS, et al.: Visualizing ribosome biogenesis: parallel assembly pathways for the 30S subunit. Science 2010, 330(6004):673-7.
• [54]Champney WS: Bacterial ribosomal subunit assembly is an antibiotic target. Curr Top Med Chem 2003, 3(9):929-47.
• [55]Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E: A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337(6096):816-21.
• [56]Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al.: RNA-guided human genome engineering via Cas9. Science 2013, 339(6121):823-6.
• [57]Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al.: Multiplex genome engineering using CRISPR/Cas systems. Science 2013, 339(6121):819-23.
• [58]Loreni F, Mancino M, Biffo S: Translation factors and ribosomal proteins control tumor onset and progression: how? Oncogene 2014, 33(17):2145-56.
• [59]Amann E, Ochs B, Abel KJ: Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 1988, 69(2):301-15.
• [60]Hanahan D: Studies on transformation of Escherichia coli with plasmids. J Mol Biol 1983, 166(4):557-80.
• [61]Cherepanov PP, Wackernagel W: Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 1995, 158(1):9-14.