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Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G.

Mandava CS, Peisker K, Ederth J, Kumar R, Ge X, Szaflarski W, Sanyal S - Nucleic Acids Res. (2011)

Bottom Line: Thus JE105 harbors ribosomes with only a single L12 dimer.When tested in a cell-free reconstituted transcription-translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits.Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Uppsala University, BMC, Box-596, SE-751 24 Uppsala, Sweden.

ABSTRACT
The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dimers that bind to the adjacent sites on protein L10, spanning 10 amino acids each from the L10 C-terminus. To study why multiple L12 dimers are required on the ribosome, we created a chromosomally engineered Escherichia coli strain, JE105, in which the peripheral L12 dimer binding site was deleted. Thus JE105 harbors ribosomes with only a single L12 dimer. Compared to MG1655, the parental strain with two L12 dimers, JE105 showed significant growth defect suggesting suboptimal function of the ribosomes with one L12 dimer. When tested in a cell-free reconstituted transcription-translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits. Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G. Varying number of L12 dimers on the ribosome can be a mechanism in bacteria for modulating the rate of translation in response to growth condition.

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In vitro synthesis of firefly luciferase. Synthesis of full-length, active firefly luciferase followed by the increase in luminescence in a reconstituted transcription–translation system with 70S ribosomes (a) and ribosomal subunits (b) from MG1655, MRE600 (both containing two L12 dimers) and JE105 (single L12 dimer).
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gkr1031-F3: In vitro synthesis of firefly luciferase. Synthesis of full-length, active firefly luciferase followed by the increase in luminescence in a reconstituted transcription–translation system with 70S ribosomes (a) and ribosomal subunits (b) from MG1655, MRE600 (both containing two L12 dimers) and JE105 (single L12 dimer).

Mentions: JE105 ribosomes were compared with ribosomes from MG1655 and MRE600 (both of which contained two L12 dimers) for their activity in synthesis of a full-length protein firefly luciferase in vitro. For that, a cell-free transcription–translation system composed of highly active, purified components from E. coli was employed (27). JE105 ribosomes were notably slower in luciferase synthesis than the other two ribosomes (Figure 3a). Further, when ribosomal subunits from these strains were mixed in pair-wise combinations, the reactions with JE105 50S subunits showed slower increase in luminescence irrespective of the source of the 30S subunit (Figure 3b). Thus, it could be inferred that the defect in JE105 ribosomes did arise from its 50S subunit containing single L12-dimer. It is also noteworthy that the ribosomal subunits from MG1655 and MRE600 strains behaved very similarly in this assay and that the 30S subunits from JE105 were as active as those from MRE600.Figure 3.


Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G.

Mandava CS, Peisker K, Ederth J, Kumar R, Ge X, Szaflarski W, Sanyal S - Nucleic Acids Res. (2011)

In vitro synthesis of firefly luciferase. Synthesis of full-length, active firefly luciferase followed by the increase in luminescence in a reconstituted transcription–translation system with 70S ribosomes (a) and ribosomal subunits (b) from MG1655, MRE600 (both containing two L12 dimers) and JE105 (single L12 dimer).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299993&req=5

gkr1031-F3: In vitro synthesis of firefly luciferase. Synthesis of full-length, active firefly luciferase followed by the increase in luminescence in a reconstituted transcription–translation system with 70S ribosomes (a) and ribosomal subunits (b) from MG1655, MRE600 (both containing two L12 dimers) and JE105 (single L12 dimer).
Mentions: JE105 ribosomes were compared with ribosomes from MG1655 and MRE600 (both of which contained two L12 dimers) for their activity in synthesis of a full-length protein firefly luciferase in vitro. For that, a cell-free transcription–translation system composed of highly active, purified components from E. coli was employed (27). JE105 ribosomes were notably slower in luciferase synthesis than the other two ribosomes (Figure 3a). Further, when ribosomal subunits from these strains were mixed in pair-wise combinations, the reactions with JE105 50S subunits showed slower increase in luminescence irrespective of the source of the 30S subunit (Figure 3b). Thus, it could be inferred that the defect in JE105 ribosomes did arise from its 50S subunit containing single L12-dimer. It is also noteworthy that the ribosomal subunits from MG1655 and MRE600 strains behaved very similarly in this assay and that the 30S subunits from JE105 were as active as those from MRE600.Figure 3.

Bottom Line: Thus JE105 harbors ribosomes with only a single L12 dimer.When tested in a cell-free reconstituted transcription-translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits.Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Uppsala University, BMC, Box-596, SE-751 24 Uppsala, Sweden.

ABSTRACT
The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dimers that bind to the adjacent sites on protein L10, spanning 10 amino acids each from the L10 C-terminus. To study why multiple L12 dimers are required on the ribosome, we created a chromosomally engineered Escherichia coli strain, JE105, in which the peripheral L12 dimer binding site was deleted. Thus JE105 harbors ribosomes with only a single L12 dimer. Compared to MG1655, the parental strain with two L12 dimers, JE105 showed significant growth defect suggesting suboptimal function of the ribosomes with one L12 dimer. When tested in a cell-free reconstituted transcription-translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits. Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G. Varying number of L12 dimers on the ribosome can be a mechanism in bacteria for modulating the rate of translation in response to growth condition.

Show MeSH
Related in: MedlinePlus