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The conserved GTPase LepA contributes mainly to translation initiation in Escherichia coli - PubMed

  • ️Wed Jan 01 2014

The conserved GTPase LepA contributes mainly to translation initiation in Escherichia coli

Rohan Balakrishnan et al. Nucleic Acids Res. 2014.

Abstract

LepA is a paralog of EF-G found in all bacteria. Deletion of lepA confers no obvious growth defect in Escherichia coli, and the physiological role of LepA remains unknown. Here, we identify nine strains (ΔdksA, ΔmolR1, ΔrsgA, ΔtatB, ΔtonB, ΔtolR, ΔubiF, ΔubiG or ΔubiH) in which ΔlepA confers a synthetic growth phenotype. These strains are compromised for gene regulation, ribosome assembly, transport and/or respiration, indicating that LepA contributes to these functions in some way. We also use ribosome profiling to deduce the effects of LepA on translation. We find that loss of LepA alters the average ribosome density (ARD) for hundreds of mRNA coding regions in the cell, substantially reducing ARD in many cases. By contrast, only subtle and codon-specific changes in ribosome distribution along mRNA are seen. These data suggest that LepA contributes mainly to the initiation phase of translation. Consistent with this interpretation, the effect of LepA on ARD is related to the sequence of the Shine-Dalgarno region. Global perturbation of gene expression in the ΔlepA mutant likely explains most of its phenotypes.

© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.
Figure 1.

Examples of genes that exhibit decreased ARD in the absence of LepA. Total-RNA and ribosome-footprint read counts for WT, mutant (M) and complemented (C) strains are shown mapped back to the genome in the vicinity of ychH (A), raiA (B), lldP (C), dctA (D) and aldA (E). Ribosome-footprint reads are mapped to the genomic position corresponding to the predicted central nt of the P codon, and total-RNA reads are mapped to the center of the read fragment. Read counts are normalized with respect to total number of reads (after quality control and rRNA read removal), making the histograms of analogous data tracks directly comparable in each panel.

Figure 2.
Figure 2.

Gene expression is globally perturbed in the absence of LepA. Normalized gene-by-gene coverages are compared between ribosome footprints and total RNA in WT, mutant and complemented strains (as indicated). A measure of spread, formula image, was calculated for each of the comparisons, yielding 0.62 ± 0.02, 1.12 ± 0.02 and 0.88 ± 0.03 for the WT, M and C samples, respectively. Student's t-tests on the values of formula image in all three replicates yield P = 1.6 × 10−5 for the WT versus M, and P = 1.2 × 10−3 for the M versus C comparison, showing significant perturbation of translation efficiencies (i.e. changes in ARD values) due to loss of LepA.

Figure 3.
Figure 3.

Loss of LepA results in fewer polysomes and more free subunits. Representative A254 traces of sucrose gradient profiles of wild-type (A), ΔlepA (B) and ΔlepA(pLEPA) (C) strains, with peaks corresponding to 30S and 50S subunits, 70S monosomes and polysomes (as indicated). The percentage of polysomes, 70S monosomes, and subunits (30S plus 50S) are graphed below (mean ± SEM). Differences deemed significant are seen for polysome levels (WT versus M, P = 2.4 × 10−2; C versus M, P = 1.9 × 10−2) and subunit levels (WT versus M, P = 2.6 × 10−2), based on Student's t-test.

Figure 4.
Figure 4.

Effects of LepA on ARD are related to the TIR sequence. Nt frequencies at each position of the TIR were determined for the subset of genes with decreased ARD in the absence of LepA (WT, C > M; 237 genes), the subset of genes with increased ARD in absence of LepA (WT, C < M; 283 genes) and all genes analyzed (1870). Purine and pyrimidine frequencies for each subset, relative to those of the complete set, are plotted as a function of TIR position (as indicated; position zero corresponds to the first nt of the start codon). Binomial tests indicate that pyrimidines are significantly underrepresented in the former subset (WT, C > M) at positions −11 (P = 7.4 × 10−4), −10 (P = 2.9 × 10−3) and −9 (P = 1.6 × 10−3) and significantly overrepresented in the latter subset (WT, C < M) at positions −11 (P = 3.3 × 10−2), −10 (P = 3.7 × 10−4), −9 (P = 2.5 × 10−2) and −8 (P = 2.6 × 10−2), data points marked with asterisks. The differences seen at the first position of the start codon (position zero) are deemed less than statistically significant (only 40 of the 1870 analyzed genes begin with a pyrimidine). Genes infC and pcnB have the rare start codon AUU and hence were omitted from the analysis. The second and third nts of the start codon (UG) are otherwise invariant and assigned the value of 1.0.

Figure 5.
Figure 5.

LepA prevents ribosomal pausing at certain GGU codons. Aligned are the coding sequences corresponding to the predicted paused ribosomes seen specifically in the mutant strain. The left column identifies the gene and the codon number (of the P codon of the paused complex). Codon GGU (red) is significantly overrepresented as the A codon (P = 7.4 × 10−13), based on a two-tailed binomial test. GGC (blue), the other codon recognized by Gly-tRNAGly3, is seen to occupy the A site in two cases, which is deemed less-than-significant enrichment.

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