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

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.

Figure 1.

Construction and characterization of JE105 strain. (a) A scheme showing the steps for construction of JE105, where last 33 nucleotides of rplJ gene in the chromosome of E. coli MG1655 was replaced with an Amp cassette by means of lambda Red recombineering (left). While MG1655 ribosome has two L12 dimers bound to L10, JE105 ribosome lacked the peripheral L12 dimer due to deletion of the binding site (II) (right). (b) Agarose gel electrophoresis showing bands from colony PCR with MG1655 (lanes 3 and 5) and JE105 (lanes 2 and 4) using primers In Amp and Down L12 (lanes 2 and 3) and Up L10 and Down L12 (lanes 4 and 5). (c) The growth curves for MG1655 (black) and JE105 (red) in LB at 37°C. The generation times are listed in the box.

Figure 2.

Characterization of single L12 dimer ribosomes from JE105. (a) Native composite gel electrophoresis with MG1655 and JE105 ribosomes. (b) SDS–PAGE analysis of the isolated L8 complex from MG1655 and JE105 ribosomes. (c) Quantitative Western blot with cell lysate and total ribosomal proteins from MG1655 and JE105, using antibodies against L12 and S1 proteins. Pure L12 protein was used as a control. (d) Comparison of the ribosomal proteins from JE105 and MG1655 (in the box) in 2D-gel electrophoresis. The L12 spots are labeled with the arrow.

Figure 3.

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).

Figure 4.

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-tRNA^fMet, 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 (λ_Ex = 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).

Figure 5.

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/k_{obs (tripeptide)} − 2 (1/k_{obs (dipeptide)})].

Figure 6.

EF-G binding to the ribosome and GTP hydrolysis. Comparison of the naked 70S ribosomes from MRE600 (black trace), MG1655 (blue trace) (both contain two L12 dimers) and JE105 (red trace) (single L12 dimer) in (a) time course of binding of EF-G-rho followed by increase in rhodamine fluorescence at 590 nm and (b) in stimulation of GTP hydrolysis by EF-G measured by Pi release using MDCC-PBP.