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Translation efficiency is maintained at elevated temperature in Escherichia coli.

ABSTRACT: Cellular protein levels are dictated by the balance between gene transcription, mRNA translation, and protein degradation, among other factors. Translation requires the interplay of several RNA hybridization processes, which are expected to be temperature-sensitive. We used ribosome profiling to monitor translation in Escherichia coli at 30 °C and to investigate how this changes after 10-20 min of heat shock at 42 °C. Translation efficiencies are robustly maintained after thermal heat shock and after mimicking the heat-shock response transcriptional program at 30 °C by overexpressing the heat shock ? factor encoded by the rpoH gene. We compared translation efficiency, the ratio of ribosome footprint reads to mRNA reads for each gene, to parameters derived from gene sequences. Genes with stable mRNA structures, non-optimal codon use, and those whose gene product is cotranslationally translocated into the inner membrane are generally less highly translated than other genes. Comparison with other published datasets suggests a role for translational elongation in coupling mRNA structures to translation initiation. Genome-wide calculations of the temperature dependence of mRNA structure predict that relatively few mRNAs show a melting transition between 30 and 42 °C, consistent with the observed lack of changes in translation efficiency. We developed a linear model with six parameters that can predict 38% of the variation in translation efficiency between genes, which may be useful in interpreting transcriptome data.

SUBMITTER: Morgan GJ

PROVIDER: S-EPMC5777253 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Translation efficiency is maintained at elevated temperature in <i>Escherichia coli</i>.

Morgan Gareth J GJ Burkhardt David H DH Kelly Jeffery W JW Powers Evan T ET

The Journal of biological chemistry 20171128 3

Cellular protein levels are dictated by the balance between gene transcription, mRNA translation, and protein degradation, among other factors. Translation requires the interplay of several RNA hybridization processes, which are expected to be temperature-sensitive. We used ribosome profiling to monitor translation in <i>Escherichia coli</i> at 30 °C and to investigate how this changes after 10-20 min of heat shock at 42 °C. Translation efficiencies are robustly maintained after thermal heat sho ...[more]

PMID: 29183994

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Project description:Translational regulation was investigated at the genome-scale in Escherichia coli cells. Using the polysome profiling method, the ribosome occupancy (RO) and ribosome density (RD) of different mRNA copies were determined for several hundred mRNAs during the exponential- and stationary-phases, providing the most complete characterization of such regulation in E. coli. Although for most genes, nearly all mRNAs (>90%) were undergoing translation, they were loaded with far fewer than the theoretical maximum number of ribosomes, suggesting translation limitation at the initiation step. Multiple linear regression was used to identify key intrinsic factors involved in the genome-wide regulation of RO and RD (i.e., open reading frame GC%, protein function, and localization). Unexpectedly, mRNA concentration, a factor that depends on cell physiology, was predicted to positively regulate RO and RD during the exponential- and stationary-phases. Using a set of selected genes controlled by an inducible promoter, we confirmed that increasing the mRNA concentration upon transcription induction led to increases in both RO and ribosome load. The fact that this relationship between mRNA concentration and translation parameters was also effective when E. coli cells naturally adapted to carbon source changes demonstrates its physiological relevance. This work demonstrated that translation regulation is positively controlled by transcript availability. This new mechanism contributed to the codirectional regulation of transcription and translation with synergistic effects on gene expression and provided a systemic understanding of E. coli cell function. IMPORTANCE The process of gene expression is divided into translation and transcription. Considerable efforts have been made in bacteria to characterize the mechanisms underlying translational regulation and identify the regulatory factors for particular mRNAs. However, to understand bacterial physiology and adaptation, it is important to elucidate genome-wide translational regulation and examine its coordination with transcriptional regulation. Here, we provided a genome-wide picture of translational regulation in Escherichia coli. For most genes, nearly all mRNA copies were found to undergo translation but were loaded with a low number of ribosomes. We showed that mRNA concentration had a positive effect on translation regulation, linking translational regulation to transcriptional regulation as well as to cell physiology and growth conditions. The codirectional regulation of transcription and translation had synergistic effects on gene expression, contributing to E. coli cell function optimization. This finding could be used in biotechnology to optimize strategies for recombinant protein synthesis.

Project description:To enable accurate and efficient translation, sulfur modifications are introduced posttranscriptionally into nucleosides in tRNAs. The biosynthesis of tRNA sulfur modifications involves unique sulfur trafficking systems for the incorporation of sulfur atoms in different nucleosides of tRNA. One of the proteins that is involved in inserting the sulfur for 5-methylaminomethyl-2-thiouridine (mnm5s2U34) modifications in tRNAs is the TusA protein. TusA, however, is a versatile protein that is also involved in numerous other cellular pathways. Despite its role as a sulfur transfer protein for the 2-thiouridine formation in tRNA, a fundamental role of TusA in the general physiology of Escherichia coli has also been discovered. Poor viability, a defect in cell division, and a filamentous cell morphology have been described previously for tusA-deficient cells. In this report, we aimed to dissect the role of TusA for cell viability. We were able to show that the lack of the thiolation status of wobble uridine (U34) nucleotides present on Lys, Gln, or Glu in tRNAs has a major consequence on the translation efficiency of proteins; among the affected targets are the proteins RpoS and Fis. Both proteins are major regulatory factors, and the deregulation of their abundance consequently has a major effect on the cellular regulatory network, with one consequence being a defect in cell division by regulating the FtsZ ring formation.IMPORTANCE More than 100 different modifications are found in RNAs. One of these modifications is the mnm5s2U modification at the wobble position 34 of tRNAs for Lys, Gln, and Glu. The functional significance of U34 modifications is substantial since it restricts the conformational flexibility of the anticodon, thus providing translational fidelity. We show that in an Escherichia coli TusA mutant strain, involved in sulfur transfer for the mnm5s2U34 thio modifications, the translation efficiency of RpoS and Fis, two major cellular regulatory proteins, is altered. Therefore, in addition to the transcriptional regulation and the factors that influence protein stability, tRNA modifications that ensure the translational efficiency provide an additional crucial regulatory factor for protein synthesis.

Project description:BackgroundCampylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni.ResultsWe generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain.ConclusionsWe determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level.

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Translation efficiency is maintained at elevated temperature in Escherichia coli.

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Translation efficiency is maintained at elevated temperature in <i>Escherichia coli</i>.

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