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Conservation of bacterial protein synthesis machinery: initiation and elongation in Mycobacterium smegmatis


Bruell, C M; Eichholz, C; Kubarenko, A; Post, V; Katunin, V I; Hobbie, S N; Rodnina, M V; Böttger, E C (2008). Conservation of bacterial protein synthesis machinery: initiation and elongation in Mycobacterium smegmatis. Biochemistry, 47(34):8828-8839.

Abstract

Most of our understanding of ribosome function is based on experiments utilizing translational components from Escherichia coli. It is not clear to which extent the details of translation mechanisms derived from this single organism are true for all bacteria. Here we investigate translation factor-dependent reactions of initiation and elongation in a reconstituted translation system from a Gram-positive bacterium Mycobacterium smegmatis. This organism was chosen because mutations in rRNA have very different phenotypes in E. coli and M. smegmatis, and the docking site for translational GTPases, the L12 stalk, is extended in the ribosomes from M. smegmatis compared to E. coli. M. smegmatis genes coding for IF1, IF2, IF3, EF-G, and EF-Tu were identified by sequence alignments; the respective recombinant proteins were prepared and studied in a variety of biochemical and biophysical assays with M. smegmatis ribosomes. We found that the activities of initiation and elongation factors and the rates of elemental reactions of initiation and elongation of protein synthesis are remarkably similar with M. smegmatis and E. coli components. The data suggest a very high degree of conservation of basic translation mechanisms, probably due to coevolution of the ribosome components and translation factors. This work establishes the reconstituted translation system from individual purified M. smegmatis components as an alternative to that from E. coli to study the mechanisms of translation and to test the action of antibiotics against Gram-positive bacteria.

Most of our understanding of ribosome function is based on experiments utilizing translational components from Escherichia coli. It is not clear to which extent the details of translation mechanisms derived from this single organism are true for all bacteria. Here we investigate translation factor-dependent reactions of initiation and elongation in a reconstituted translation system from a Gram-positive bacterium Mycobacterium smegmatis. This organism was chosen because mutations in rRNA have very different phenotypes in E. coli and M. smegmatis, and the docking site for translational GTPases, the L12 stalk, is extended in the ribosomes from M. smegmatis compared to E. coli. M. smegmatis genes coding for IF1, IF2, IF3, EF-G, and EF-Tu were identified by sequence alignments; the respective recombinant proteins were prepared and studied in a variety of biochemical and biophysical assays with M. smegmatis ribosomes. We found that the activities of initiation and elongation factors and the rates of elemental reactions of initiation and elongation of protein synthesis are remarkably similar with M. smegmatis and E. coli components. The data suggest a very high degree of conservation of basic translation mechanisms, probably due to coevolution of the ribosome components and translation factors. This work establishes the reconstituted translation system from individual purified M. smegmatis components as an alternative to that from E. coli to study the mechanisms of translation and to test the action of antibiotics against Gram-positive bacteria.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Microbiology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2008
Deposited On:21 Oct 2008 15:29
Last Modified:05 Apr 2016 12:30
Publisher:American Chemical Society
ISSN:0006-2960
Publisher DOI:10.1021/bi800527k
PubMed ID:18672904

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