title: Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli.
abstract: 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.
authors: New England Biolabs, Inc., 240 County Road, Ipswich, MA, 01938, United States of America.
authors: Aashish
authors: A
authors: Srivastava
authors: New England Biolabs, Inc., 240 County Road, Ipswich, MA, 01938, United States of America.
authors: Haruichi
authors: H
authors: Asahara
authors: Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100176, China.
authors: Meng
authors: M
authors: Zhang
authors: Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100176, China.
authors: Weijia
authors: W
authors: Zhang
authors: Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100176, China.
authors: Haiying
authors: H
authors: Liu
authors: Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100176, China.
authors: Sheng
authors: S
authors: Cui
authors: Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100176, China.
authors: Qi
authors: Q
authors: Jin
authors: New England Biolabs, Inc., 240 County Road, Ipswich, MA, 01938, United States of America.
authors: Shaorong
authors: S
authors: Chong
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D009161:Mycobacterium / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology
chemical_list: D000604:Amino Acyl-tRNA Synthetases