Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Escherichia coli/*metabolism ; Mycobacterium/*metabolism; Amino Acyl-tRNA Synthetases/metabolism ; Mycobacterium smegmatis/metabolism ; Protein Transport/physiology

Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis), purified tRNAs and ribosomes from Mycobacterium smegmatis (M. smegmatis), and an aminoacyl-tRNA synthetase (AARS) mixture from the cell-extract of M. smegmatis. We demonstrate that such mycobacterial translation system was efficient in in vitro protein synthesis, and enabled functional comparisons of translational components between the gram-positive Mycobacterium and the gram-negative E. coli. Although mycobacterial translation factors and ribosomes were highly compatible with their E. coli counterparts, M. smegmatis tRNAs were not properly charged by the E. coli AARSs to allow efficient translation of a reporter. In contrast, both E. coli and M. smegmatis tRNAs exhibited similar activity with the semi-purified M. smegmatis AARSs mixture for in vitro translation. We further demonstrated the use of both mycobacterial and E. coli translation systems as comparative in vitro assays for small-molecule antibiotics that target protein translation. While mycobacterial and E. coli translation were both inhibited at the same IC50 by the antibiotic spectinomycin, mycobacterial translation was preferentially inhibited by the antibiotic tetracycline, suggesting that there may be structural differences at the antibiotic binding sites between the ribosomes of Mycobacterium and E. coli. Our results illustrate an alternative approach for antibiotic discovery and functional studies of protein translation in mycobacteria and possibly other bacterial pathogens.
Competing Interests: Our affiliation with New England Biolabs does not alter our adherence to PLOS ONE policies on sharing data and materials.

J Antimicrob Chemother. 2007 Mar;59(3):544-7. (PMID: 17242035)
Biochem Biophys Res Commun. 2007 Jan 5;352(1):270-6. (PMID: 17113037)
Nat Biotechnol. 2001 Aug;19(8):751-5. (PMID: 11479568)
J Infect Dis. 2015 Apr 1;211 Suppl 2:S21-8. (PMID: 25765103)
Nucleic Acids Res. 2010 Jul;38(13):e141. (PMID: 20457746)
Proc Natl Acad Sci U S A. 2014 Jan 21;111(3):1132-7. (PMID: 24395793)
Biochemistry. 2008 Aug 26;47(34):8828-39. (PMID: 18672904)
Science. 2011 Sep 16;333(6049):1630-2. (PMID: 21835980)
Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7877-82. (PMID: 12788972)
N Engl J Med. 2012 Sep 6;367(10):931-6. (PMID: 22931261)
ACS Chem Biol. 2007 Aug 17;2(8):545-52. (PMID: 17696316)
Drug Discov Today. 2006 Jan;11(1-2):21-7. (PMID: 16478687)
Proc Natl Acad Sci U S A. 2010 Oct 5;107(40):17152-7. (PMID: 20876128)
J Am Chem Soc. 2014 Oct 8;136(40):14031-8. (PMID: 25188838)
Nucleic Acids Res. 2012 Sep;40(16):7932-45. (PMID: 22723376)
Proc Natl Acad Sci U S A. 2010 Oct 5;107(40):17158-63. (PMID: 20876130)
Science. 2001 Jan 19;291(5503):498-501. (PMID: 11228145)
J Mol Biol. 2008 Mar 28;377(3):831-44. (PMID: 18279892)
EMBO J. 2001 Apr 17;20(8):1829-39. (PMID: 11296217)
Proc Natl Acad Sci U S A. 2011 Apr 12;108(15):6073-8. (PMID: 21383133)
PLoS One. 2012;7(2):e31742. (PMID: 22384065)

EC 6.1.1.- (Amino Acyl-tRNA Synthetases)

Date Created: 20160827 Date Completed: 20170818 Latest Revision: 20181113

20231215

PMC5001721

10.1371/journal.pone.0162020

27564552