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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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Related in: MedlinePlus

Dipeptide and tripeptide formation experiments; scheme of translation elongation. Comparison of the MRE600 (black trace), MG1655 (blue trace) (both contain two L12 dimers) and JE105 (red trace) (single L12 dimer) ribosomes in dipeptide (a) and tripeptide (b) formation assays. (c) Schematic representation of different steps of elongation showing the average time analysis for by MG1655/MRE600 (in black) and JE105 (in red) ribosomes. The average time for EF-G driven steps was estimated as [1/kobs (tripeptide) − 2 (1/kobs (dipeptide))].
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gkr1031-F5: Dipeptide and tripeptide formation experiments; scheme of translation elongation. Comparison of the MRE600 (black trace), MG1655 (blue trace) (both contain two L12 dimers) and JE105 (red trace) (single L12 dimer) ribosomes in dipeptide (a) and tripeptide (b) formation assays. (c) Schematic representation of different steps of elongation showing the average time analysis for by MG1655/MRE600 (in black) and JE105 (in red) ribosomes. The average time for EF-G driven steps was estimated as [1/kobs (tripeptide) − 2 (1/kobs (dipeptide))].

Mentions: We have tested the single L12 dimer ribosomes from JE105 in the steps of elongation by monitoring the rate of formation of fMet-Leu dipeptide and fMet-Leu-Leu tripeptide starting from a complete 70S initiation complex (70S IC). The dipeptide experiment essentially integrated the steps starting from EF-Tu-Leu-tRNALeu·GTP binding, GTP hydrolysis and release of EF-Tu·GDP and Pi (referred as EF-Tu mediated steps) to the peptide bond formation. The rate of dipeptide formation with 70S IC from JE105 was about the same as with that from MG1655 and MRE600 (kobs = 51 ± 7 s−1) (Figure 5a). Although somewhat surprising, this result suggested no necessity of multiple L12 dimers in the steps involving EF-Tu. It also indicates that peptidyl transfer is independent of the number of L12 proteins on the ribosome.Figure 5.


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)

Dipeptide and tripeptide formation experiments; scheme of translation elongation. Comparison of the MRE600 (black trace), MG1655 (blue trace) (both contain two L12 dimers) and JE105 (red trace) (single L12 dimer) ribosomes in dipeptide (a) and tripeptide (b) formation assays. (c) Schematic representation of different steps of elongation showing the average time analysis for by MG1655/MRE600 (in black) and JE105 (in red) ribosomes. The average time for EF-G driven steps was estimated as [1/kobs (tripeptide) − 2 (1/kobs (dipeptide))].
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1031-F5: Dipeptide and tripeptide formation experiments; scheme of translation elongation. Comparison of the MRE600 (black trace), MG1655 (blue trace) (both contain two L12 dimers) and JE105 (red trace) (single L12 dimer) ribosomes in dipeptide (a) and tripeptide (b) formation assays. (c) Schematic representation of different steps of elongation showing the average time analysis for by MG1655/MRE600 (in black) and JE105 (in red) ribosomes. The average time for EF-G driven steps was estimated as [1/kobs (tripeptide) − 2 (1/kobs (dipeptide))].
Mentions: We have tested the single L12 dimer ribosomes from JE105 in the steps of elongation by monitoring the rate of formation of fMet-Leu dipeptide and fMet-Leu-Leu tripeptide starting from a complete 70S initiation complex (70S IC). The dipeptide experiment essentially integrated the steps starting from EF-Tu-Leu-tRNALeu·GTP binding, GTP hydrolysis and release of EF-Tu·GDP and Pi (referred as EF-Tu mediated steps) to the peptide bond formation. The rate of dipeptide formation with 70S IC from JE105 was about the same as with that from MG1655 and MRE600 (kobs = 51 ± 7 s−1) (Figure 5a). Although somewhat surprising, this result suggested no necessity of multiple L12 dimers in the steps involving EF-Tu. It also indicates that peptidyl transfer is independent of the number of L12 proteins on the ribosome.Figure 5.

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