Conservation of bacterial protein synthesis machinery: initiation and elongation in Mycobacterium smegmatis.
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.
Bruell
Christian M
CM
Institute of Medical Microbiology, University of Zurich, Switzerland.
Eichholz
Carolin
C
Kubarenko
Andriy
A
Post
Virginia
V
Katunin
Vladimir I
VI
Hobbie
Sven N
SN
Rodnina
Marina V
MV
Böttger
Erik C
EC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism*; D020653:Peptide Elongation Factor G / Q000235:genetics / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D039861:Prokaryotic Initiation Factor-1 / Q000235:genetics / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism; D039665:Prokaryotic Initiation Factor-3 / Q000235:genetics / Q000378:metabolism; D039662:Prokaryotic Initiation Factors; D011485:Protein Binding; D014176:Protein Biosynthesis; D012360:RNA, Transfer, Phe / Q000378:metabolism; D054657:Ribosome Subunits / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D039861:Prokaryotic Initiation Factor-1; D039881:Prokaryotic Initiation Factor-2; D039665:Prokaryotic Initiation Factor-3; D039662:Prokaryotic Initiation Factors; D012360:RNA, Transfer, Phe; D010444:Peptide Elongation Factor Tu