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Translation Elongation Factor 4 (LepA) Contributes to Tetracycline Susceptibility by Stalling Elongating Ribosomes.

ABSTRACT: Even though elongation factor 4 (EF4) is the third most conserved protein in bacteria, its physiological functions remain largely unknown and its proposed molecular mechanisms are conflicting among previous studies. In the present study, we show that the growth of an Escherichia coli strain is more susceptible to tetracycline than its EF4 knockout strain. Consistent with previous studies, our results suggested that EF4 affects ribosome biogenesis when tetracycline is present. Through ribosome profiling analysis, we discovered that EF4 causes 1-nucleotide shifting of ribosomal footprints on mRNA when cells have been exposed to tetracycline. In addition, when tetracycline is present, EF4 inhibits the elongation of protein synthesis, which leads to the accumulation of ribosomes in the early segment of mRNA. Altogether, when cells are exposed to tetracycline, EF4 alters both ribosome biogenesis and the elongation phase of protein synthesis.

SUBMITTER: Liu B

PROVIDER: S-EPMC6105825 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Translation Elongation Factor 4 (LepA) Contributes to Tetracycline Susceptibility by Stalling Elongating Ribosomes.

Liu Bin B Chen Chunlai C

Antimicrobial agents and chemotherapy 20180727 8

Even though elongation factor 4 (EF4) is the third most conserved protein in bacteria, its physiological functions remain largely unknown and its proposed molecular mechanisms are conflicting among previous studies. In the present study, we show that the growth of an <i>Escherichia coli</i> strain is more susceptible to tetracycline than its EF4 knockout strain. Consistent with previous studies, our results suggested that EF4 affects ribosome biogenesis when tetracycline is present. Through ribo ...[more]

PMID: 29784847

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Translation elongation factor 4 (LepA) contributes to tetracycline susceptibility by stalling elongating ribosomes

Project description:Purpose: We use the ribosome profiling protocol to understand EF4 mediated translation events. Methods: We used ribosome profiling data to analyze by plastid software. The ribosome were purified by sucrose gradient separation. Results: Using sequencing data, we found that EF4 mediates the 1-nucleotide conformational change of ribosomes. We also found that many genes involved in translation after tetracycline treatment. Finally, we found that EF4 stalls the elongating ribosomes globally. Conclusions: Our results suggest that EF4 mediates both 30S biogenesis and translation elongation process, in particular, EF4 stalls the elongating ribosomes globally.

2018-05-14 | GSE106448 | GEO

Translation elongation factor 4 (LepA) contributes to tetracycline susceptibility by stalling elongating ribosomes

Project description:Translation elongation factor 4 (LepA) contributes to tetracycline susceptibility by stalling elongating ribosomes

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

Project description: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.

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RPL3L-containing ribosomes determine translation elongation dynamics required for cardiac function.

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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

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DksA guards elongating RNA polymerase against ribosome-stalling-induced arrest.

Project description:In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.

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