Implications of Macromolecular Crowding and Reducing Conditions for in Vitro Ribosome Construction

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2015 Apr 22

PMID 25897121

Citations 20

Authors

Brian R Fritz

Osman K Jamil

Michael C Jewett

Affiliations

Soon will be listed here.

Abstract

In vitro construction of Escherichia coli ribosomes could elucidate a deeper understanding of these complex molecular machines and make possible the production of synthetic variants with new functions. Toward this goal, we recently developed an integrated synthesis, assembly and translation (iSAT) system that allows for co-activation of ribosomal RNA (rRNA) transcription and ribosome assembly, mRNA transcription and protein translation without intact cells. Here, we discovered that macromolecular crowding and reducing agents increase overall iSAT protein synthesis; the combination of 6% w/v Ficoll 400 and 2 mM DTBA yielded approximately a five-fold increase in overall iSAT protein synthesis activity. By utilizing a fluorescent RNA aptamer, fluorescent reporter proteins and ribosome sedimentation analysis, we showed that crowding agents increase iSAT yields by enhancing translation while reducing agents increase rRNA transcription and ribosome assembly. Finally, we showed that iSAT ribosomes possess ∼70% of the protein synthesis activity of in vivo-assembled E. coli ribosomes. This work improves iSAT protein synthesis through the addition of crowding and reducing agents, provides a thorough understanding of the effect of these additives within the iSAT system and demonstrates how iSAT allows for manipulation and analysis of ribosome biogenesis in the context of an in vitro transcription-translation system.

Citing Articles

Autonomous ribosome biogenesis in vitro.

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References

Agalarov S, Williamson J . A hierarchy of RNA subdomains in assembly of the central domain of the 30 S ribosomal subunit. RNA. 2000; 6(3):402-8. PMC: 1369922. DOI: 10.1017/s1355838200991945. View

Liu Y, Fritz B, Anderson M, Schoborg J, Jewett M . Characterizing and alleviating substrate limitations for improved in vitro ribosome construction. ACS Synth Biol. 2014; 4(4):454-62. DOI: 10.1021/sb5002467. View

Semrad K, Green R . Osmolytes stimulate the reconstitution of functional 50S ribosomes from in vitro transcripts of Escherichia coli 23S rRNA. RNA. 2002; 8(4):401-11. PMC: 1370264. DOI: 10.1017/s1355838202029722. View

Ogle J, Carter A, Ramakrishnan V . Insights into the decoding mechanism from recent ribosome structures. Trends Biochem Sci. 2003; 28(5):259-66. DOI: 10.1016/S0968-0004(03)00066-5. View

Kim D, Swartz J . Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotechnol Bioeng. 2004; 85(2):122-9. DOI: 10.1002/bit.10865. View

Jewett M, Swartz J . Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnol Bioeng. 2004; 86(1):19-26. DOI: 10.1002/bit.20026. View

Yin G, Swartz J . Enhancing multiple disulfide bonded protein folding in a cell-free system. Biotechnol Bioeng. 2004; 86(2):188-95. DOI: 10.1002/bit.10827. View

Swartz J, Jewett M, Woodrow K . Cell-free protein synthesis with prokaryotic combined transcription-translation. Methods Mol Biol. 2004; 267:169-82. DOI: 10.1385/1-59259-774-2:169. 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

10.

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

11.

Herold M, Nierhaus K . Incorporation of six additional proteins to complete the assembly map of the 50 S subunit from Escherichia coli ribosomes. J Biol Chem. 1987; 262(18):8826-33. View

12.

Parker J . Errors and alternatives in reading the universal genetic code. Microbiol Rev. 1989; 53(3):273-98. PMC: 372737. DOI: 10.1128/mr.53.3.273-298.1989. View

13.

Cayley S, Lewis B, Guttman H, Record Jr M . Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo. J Mol Biol. 1991; 222(2):281-300. DOI: 10.1016/0022-2836(91)90212-o. View

14.

Cunningham P, Richard R, Weitzmann C, Nurse K, OFENGAND J . The absence of modified nucleotides affects both in vitro assembly and in vitro function of the 30S ribosomal subunit of Escherichia coli. Biochimie. 1991; 73(6):789-96. DOI: 10.1016/0300-9084(91)90058-9. View

15.

Hwang C, Sinskey A, Lodish H . Oxidized redox state of glutathione in the endoplasmic reticulum. Science. 1992; 257(5076):1496-502. DOI: 10.1126/science.1523409. View

16.

Derman A, Prinz W, Belin D, Beckwith J . Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science. 1993; 262(5140):1744-7. DOI: 10.1126/science.8259521. View

17.

Nakano H, Tanaka T, Kawarasaki Y, Yamane T . An increased rate of cell-free protein synthesis by condensing wheat-germ extract with ultrafiltration membranes. Biosci Biotechnol Biochem. 1994; 58(4):631-4. DOI: 10.1271/bbb.58.631. View

18.

Kim D, Kigawa T, Choi C, Yokoyama S . A highly efficient cell-free protein synthesis system from Escherichia coli. Eur J Biochem. 1996; 239(3):881-6. DOI: 10.1111/j.1432-1033.1996.0881u.x. View

19.

Green R, Noller H . In vitro complementation analysis localizes 23S rRNA posttranscriptional modifications that are required for Escherichia coli 50S ribosomal subunit assembly and function. RNA. 1996; 2(10):1011-21. PMC: 1369433. View

20.

Green R, Noller H . Reconstitution of functional 50S ribosomes from in vitro transcripts of Bacillus stearothermophilus 23S rRNA. Biochemistry. 1999; 38(6):1772-9. DOI: 10.1021/bi982246a. View