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. 2010 Jul 9;39(1):110-20.
doi: 10.1016/j.molcel.2010.06.009.

Hyperaccurate and error-prone ribosomes exploit distinct mechanisms during tRNA selection

Hani S Zaher  1 , Rachel Green
Affiliations

Hyperaccurate and error-prone ribosomes exploit distinct mechanisms during tRNA selection

Hani S Zaher et al. Mol Cell. .

Abstract

Escherichia coli strains displaying hyperaccurate (restrictive) and ribosomal ambiguity (ram) phenotypes have long been associated with alterations in rpsL and rpsD/rpsE, respectively. Crystallographic evidence shows the ribosomal proteins S12 and S4/S5 (corresponding to these genes) to be located in separate regions of the small ribosomal subunit that are important for domain closure thought to take place during tRNA selection. Mechanistically, the process of tRNA selection is separated into two distinct phases, initial selection and proofreading. Here, we explore the effects of mutations in rpsL and rpsD on these steps. Surprisingly, both restrictive and ram ribosomes primarily affect tRNA selection through alteration of the off rates of the near-cognate tRNA species but during distinct phases of the overall process (proofreading and initial selection, respectively). These observations suggest that closure interfaces (S12/h27/h44 versus S4/S5) in two distinct regions of the small ribosomal subunit function independently to promote high-fidelity tRNA selection.

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Figures

Figure 1
Figure 1. Simplified kinetic scheme of the tRNA selection pathway
Only the steps that govern discrimination are shown. The pathway is divided into two phases, initial selection and proofreading, separated by the GTPase activation step. Steps accelerated for cognate tRNAs are depicted by green arrows, whereas steps that are slowed down for near-cognate tRNAs are depicted by red arrows.
Figure 2
Figure 2. Global conformational change (“domain closure”) in the small ribosomal subunit
(A) The crystal structure of cognate anticodon stem loop (ASL)-bound 30S subunit (PDB 1IBM, r-proteins and rRNA are shown in green and orange, respectively) was superimposed on a near-cognate ASL-bound 30S subunit (PDB 1N34, shown in light gray) (Ogle et al., 2002). ASL, mRNA, S4, S5 and S12 of the cognate ASL-bound structure are depicted in yellow, pink, magenta, blue and red, respectively. As a result of cognate ASL binding, the shoulder and head domains of the subunit rotate towards the center of the subunit, where decoding takes place. For instance, regions of the 16S rRNA in the beak domain move closer to the shoulder domain (gray to orange) when the A-site is occupied with a cognate ASL. (B) Close-up of the S12/h44/h27 interface is shown, with emphasis on the decoding center, where key universally conserved nucleotides undergo conformational changes (Light blue in the presence of near-cognate ASL to dark blue in the presence of cognate ASL). Ribosomal protein S12 also moves toward the decoding center upon cognate ASL-binding. Note the proximity of the rpsL141 mutation (K42N) to the ASL-mRNA helix and to residue C912 of 16S rRNA. (C) Close-up of the S4/S5 interface, in particular the location of the S4 deletion (C-terminal helix) found in the rpsD12 mutant.
Figure 3
Figure 3. The rpsD(ram) and rpsL(res) variants exhibit the expected phenotype both in vivo and in vitro
(A) The relative luminescence of firefly luciferase (F-Luc) active site (K529) variants to that of control renilla-luciferase (R-Luc) expressed in the indicated strains is plotted (normalized to K529 F-Luc value in the WT strain) (Kramer and Farabaugh, 2007). When the K529 codon (AAA) is replaced by the non-cognate phe codon (UUU), only background activity of F-Luc is observed in all strains. In contrast, when this codon is replaced by the near-cognate codon (AAU), activity associated with the error frequency of tRNALys reading the substituted AAU codon is observed. (B) The error frequency of WT, rpsL(res) and rpsD(ram) ribosome variants in our in vitro system. The error frequency was determined on UUC programmed ribosomes with excess ternary complex containing equimolar concentrations of Phe-tRNAPhe and Leu-tRNA2Leu where the relative amounts of fMet-Leu and fMet-Phe dipeptide were determined. In both graphs, the mean of three independent experiments is plotted and the error bars represent standard deviations from the mean.
Figure 4
Figure 4. Kinetic parameters for near-cognate aa-tRNAs on variant ribosomes
(A) Determination of apparent k2 and relative amount of aa-tRNA that goes through initial selection by monitoring fluorescence changes in Leu-tRNA2Leu (Prf16/17/20) on each of three ribosomal variants. Ternary complex containing labeled aa-tRNA (0.15 μM after mixing) was rapidly mixed with ICs (1 μM after mixing) displaying the Phe UUC codon in the A site. The resulting change in fluorescence signal was fit to double exponential kinetics (smooth lines). (B) Determination of k-2. The time courses were obtained by first generating codon-recognition complexes with the GTPase-deficient EFTu H84A (0.3 μM after mixing) and then rapidly mixing it with a 10-fold excess of ternary complex containing unlabeled Phe-tRNAPhe. (C) Time courses of GTP hydrolysis with the ternary complex EFTu:GTP:Leu-tRNA2Leu with 1 μM ICs. (D) Time courses of dipeptide formation. Ternary complex was added to a final concentration of 0.5 μM (thus the maximal end-point is 0.5).
Figure 5
Figure 5. Effects of the ribosomal mutations on the rates of peptide release
Dipeptidyl-tRNA RNCs carrying f-Met-Lys-tRNALys (∼200 nM) in the P site, and displaying the indicated A-site codon were reacted with RF2 - 10 μM for the stop-codon (UGA) complex, and 100 μM for the near-stop (GAA) and non-stop (GAU) complexes. Rates plotted are the average of two independent measurements, obtained from single exponential fitting of time courses. The error bars represent the standard deviation from the mean.
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References

    1. Andersson DI, Bohman K, Isaksson LA, Kurland CG. Translation rates and misreading characteristics of rpsD mutants in Escherichia coli. Mol Gen Genet. 1982;187:467–472. - PubMed
    1. Andersson DI, Kurland CG. Ram ribosomes are defective proofreaders. Mol Gen Genet. 1983;191:378–381. - PubMed
    1. Bilgin N, Claesens F, Pahverk H, Ehrenberg M. Kinetic properties of Escherichia coli ribosomes with altered forms of S12. J Mol Biol. 1992;224:1011–1027. - PubMed
    1. Birge EA, Kurland CG. Reversion of a streptomycin-dependent strain of Escherichia coli. Mol Gen Genet. 1970;109:356–369. - PubMed
    1. Bjorkman J, Samuelsson P, Andersson DI, Hughes D. Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium. Mol Microbiol. 1999;31:53–58. - PubMed

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