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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

Fast kinetics measurements of the steps of initiation. The time course of association of MRE600 (black trace), MG1655 (blue trace) (both with two L12 dimers) and JE105 (red trace) (with single L12 dimer) 50S subunits with naked 30S subunits (a), or with 30S preIC containing mRNA, fMet-tRNAfMet, IF1 and IF2·GTP (b) followed in stopped-flow by monitoring increase in light scattering at 430 nm. The insert in (b) shows the same reaction for prolonged time. (c) The plots showing linear dependence of the observed rates of subunit association on 50S concentration in reactions with 30S preIC. (d) Single round Pi release with MRE600 (black trace) and JE105 (red trace) ribosomes, measured in parallel to subunit association in the same reaction as in (b). The increase in MDCC fluorescence upon Pi binding to PBP-MDCC was monitored at 464 nm (λEx = 425 nm) (e) Schematic representation of translation initiation where average times for individual steps were estimated for two L12 dimer MRE600 (in black) and single L12 dimer JE105 (in red) ribosomes (see ‘Materials and Methods’ section for details).
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gkr1031-F4: Fast kinetics measurements of the steps of initiation. The time course of association of MRE600 (black trace), MG1655 (blue trace) (both with two L12 dimers) and JE105 (red trace) (with single L12 dimer) 50S subunits with naked 30S subunits (a), or with 30S preIC containing mRNA, fMet-tRNAfMet, IF1 and IF2·GTP (b) followed in stopped-flow by monitoring increase in light scattering at 430 nm. The insert in (b) shows the same reaction for prolonged time. (c) The plots showing linear dependence of the observed rates of subunit association on 50S concentration in reactions with 30S preIC. (d) Single round Pi release with MRE600 (black trace) and JE105 (red trace) ribosomes, measured in parallel to subunit association in the same reaction as in (b). The increase in MDCC fluorescence upon Pi binding to PBP-MDCC was monitored at 464 nm (λEx = 425 nm) (e) Schematic representation of translation initiation where average times for individual steps were estimated for two L12 dimer MRE600 (in black) and single L12 dimer JE105 (in red) ribosomes (see ‘Materials and Methods’ section for details).

Mentions: Subunit association—The ribosomal subunits from JE105 were compared with those from MG1655 and MRE600 in the subunit association assay followed by Rayleigh light-scattering (34). When naked 50S and 30S subunits were mixed rapidly in a stopped-flow instrument, no significant difference in the rate of association was seen between the two strains (Figure 4a) suggesting that the presence of single or double L12 dimers on the 50S subunit was immaterial in the context of factor-free subunit association. This was expected from our previous work, where even a complete depletion of L12 proteins from the 50S subunits did not change the rate of association of the naked subunits (27). However, when subunit association was performed with a proper 30S pre-initiation complex programmed with mRNA, initiator tRNA and the initiation factors IF1 and IF2·GTP, the single L12 dimer containing JE105 50S subunits associated two times slower (Ka = 72 ± 5 µM−1s−1) than the MG1655 and MRE600 50S subunits containing two L12 dimers (Ka = 160 ± 5 µM−1s−1) (Figure 4b and Table 1). Further, 50S subunits from MRE600 and JE105 were titrated in the same assay and the linearly increasing observed rate of association was plotted against the 50S concentration. As shown in Figure 4c, at the higher concentration of 50S subunits, the incompetence of the single L12 dimer 50S in IF2-mediated subunit association was more pronounced in comparison with the double L12 dimer 50S. The role of L12 protein in IF2-mediated subunit association is a rather recent finding (27). Our present result strengthens that notion and suggests that multiple L12 dimers are required on the 50S subunit for proper interaction with IF2·GTP on the 30S pre-initiation complex, a key-step for rapid formation of the 70S initiation complex. It should be noted that both the titration curves with MRE600 and JE105 50S subunits passed through the origin, indicating that the samples were free from any kind of heterogeneity.Figure 4.


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)

Fast kinetics measurements of the steps of initiation. The time course of association of MRE600 (black trace), MG1655 (blue trace) (both with two L12 dimers) and JE105 (red trace) (with single L12 dimer) 50S subunits with naked 30S subunits (a), or with 30S preIC containing mRNA, fMet-tRNAfMet, IF1 and IF2·GTP (b) followed in stopped-flow by monitoring increase in light scattering at 430 nm. The insert in (b) shows the same reaction for prolonged time. (c) The plots showing linear dependence of the observed rates of subunit association on 50S concentration in reactions with 30S preIC. (d) Single round Pi release with MRE600 (black trace) and JE105 (red trace) ribosomes, measured in parallel to subunit association in the same reaction as in (b). The increase in MDCC fluorescence upon Pi binding to PBP-MDCC was monitored at 464 nm (λEx = 425 nm) (e) Schematic representation of translation initiation where average times for individual steps were estimated for two L12 dimer MRE600 (in black) and single L12 dimer JE105 (in red) ribosomes (see ‘Materials and Methods’ section for details).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1031-F4: Fast kinetics measurements of the steps of initiation. The time course of association of MRE600 (black trace), MG1655 (blue trace) (both with two L12 dimers) and JE105 (red trace) (with single L12 dimer) 50S subunits with naked 30S subunits (a), or with 30S preIC containing mRNA, fMet-tRNAfMet, IF1 and IF2·GTP (b) followed in stopped-flow by monitoring increase in light scattering at 430 nm. The insert in (b) shows the same reaction for prolonged time. (c) The plots showing linear dependence of the observed rates of subunit association on 50S concentration in reactions with 30S preIC. (d) Single round Pi release with MRE600 (black trace) and JE105 (red trace) ribosomes, measured in parallel to subunit association in the same reaction as in (b). The increase in MDCC fluorescence upon Pi binding to PBP-MDCC was monitored at 464 nm (λEx = 425 nm) (e) Schematic representation of translation initiation where average times for individual steps were estimated for two L12 dimer MRE600 (in black) and single L12 dimer JE105 (in red) ribosomes (see ‘Materials and Methods’ section for details).
Mentions: Subunit association—The ribosomal subunits from JE105 were compared with those from MG1655 and MRE600 in the subunit association assay followed by Rayleigh light-scattering (34). When naked 50S and 30S subunits were mixed rapidly in a stopped-flow instrument, no significant difference in the rate of association was seen between the two strains (Figure 4a) suggesting that the presence of single or double L12 dimers on the 50S subunit was immaterial in the context of factor-free subunit association. This was expected from our previous work, where even a complete depletion of L12 proteins from the 50S subunits did not change the rate of association of the naked subunits (27). However, when subunit association was performed with a proper 30S pre-initiation complex programmed with mRNA, initiator tRNA and the initiation factors IF1 and IF2·GTP, the single L12 dimer containing JE105 50S subunits associated two times slower (Ka = 72 ± 5 µM−1s−1) than the MG1655 and MRE600 50S subunits containing two L12 dimers (Ka = 160 ± 5 µM−1s−1) (Figure 4b and Table 1). Further, 50S subunits from MRE600 and JE105 were titrated in the same assay and the linearly increasing observed rate of association was plotted against the 50S concentration. As shown in Figure 4c, at the higher concentration of 50S subunits, the incompetence of the single L12 dimer 50S in IF2-mediated subunit association was more pronounced in comparison with the double L12 dimer 50S. The role of L12 protein in IF2-mediated subunit association is a rather recent finding (27). Our present result strengthens that notion and suggests that multiple L12 dimers are required on the 50S subunit for proper interaction with IF2·GTP on the 30S pre-initiation complex, a key-step for rapid formation of the 70S initiation complex. It should be noted that both the titration curves with MRE600 and JE105 50S subunits passed through the origin, indicating that the samples were free from any kind of heterogeneity.Figure 4.

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