In Vitro Integration of Ribosomal RNA Synthesis, Ribosome Assembly, and Translation

Overview

Journal Mol Syst Biol

Specialty Molecular Biology

Date 2013 Jun 27

PMID 23799452

Citations 77

Authors

Michael C Jewett

Brian R Fritz

Laura E Timmerman

George M Church

Affiliations

Soon will be listed here.

Abstract

Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties.

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References

Mulder A, Yoshioka C, Beck A, Bunner A, Milligan R, Potter C . Visualizing ribosome biogenesis: parallel assembly pathways for the 30S subunit. Science. 2010; 330(6004):673-7. PMC: 2990404. DOI: 10.1126/science.1193220. View

Jewett M, Swartz J . Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm. Biotechnol Bioeng. 2004; 87(4):465-72. DOI: 10.1002/bit.20139. View

Carlson E, Gan R, Hodgman C, Jewett M . Cell-free protein synthesis: applications come of age. Biotechnol Adv. 2011; 30(5):1185-94. PMC: 4038126. DOI: 10.1016/j.biotechadv.2011.09.016. View

Maki J, Culver G . Recent developments in factor-facilitated ribosome assembly. Methods. 2005; 36(3):313-20. DOI: 10.1016/j.ymeth.2005.04.008. 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

Jelenc P, Kurland C . Nucleoside triphosphate regeneration decreases the frequency of translation errors. Proc Natl Acad Sci U S A. 1979; 76(7):3174-8. PMC: 383786. DOI: 10.1073/pnas.76.7.3174. View

Weitzmann C, Cunningham P, OFENGAND J . Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA. Nucleic Acids Res. 1990; 18(12):3515-20. PMC: 331005. DOI: 10.1093/nar/18.12.3515. View

Jewett M, Forster A . Update on designing and building minimal cells. Curr Opin Biotechnol. 2010; 21(5):697-703. PMC: 2952674. DOI: 10.1016/j.copbio.2010.06.008. View

Gromadski K, Rodnina M . Kinetic determinants of high-fidelity tRNA discrimination on the ribosome. Mol Cell. 2004; 13(2):191-200. DOI: 10.1016/s1097-2765(04)00005-x. View

10.

Forster A, Church G . Towards synthesis of a minimal cell. Mol Syst Biol. 2006; 2:45. PMC: 1681520. DOI: 10.1038/msb4100090. View

11.

Isaacs F, Dwyer D, Ding C, Pervouchine D, Cantor C, Collins J . Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. 2004; 22(7):841-7. DOI: 10.1038/nbt986. View

12.

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

13.

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

14.

Hodgman C, Jewett M . Cell-free synthetic biology: thinking outside the cell. Metab Eng. 2011; 14(3):261-9. PMC: 3322310. DOI: 10.1016/j.ymben.2011.09.002. View

15.

Krzyzosiak W, Denman R, Nurse K, Hellmann W, Boublik M, GEHRKE C . In vitro synthesis of 16S ribosomal RNA containing single base changes and assembly into a functional 30S ribosome. Biochemistry. 1987; 26(8):2353-64. DOI: 10.1021/bi00382a042. 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.

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

18.

Khaitovich P, Tenson T, KLOSS P, Mankin A . Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA. Biochemistry. 1999; 38(6):1780-8. DOI: 10.1021/bi9822473. View

19.

Blanchard S, Kim H, Gonzalez Jr R, Puglisi J, Chu S . tRNA dynamics on the ribosome during translation. Proc Natl Acad Sci U S A. 2004; 101(35):12893-8. PMC: 516491. DOI: 10.1073/pnas.0403884101. View

20.

Erlacher M, Chirkova A, Voegele P, Polacek N . Generation of chemically engineered ribosomes for atomic mutagenesis studies on protein biosynthesis. Nat Protoc. 2011; 6(5):580-92. DOI: 10.1038/nprot.2011.306. View