Improving the Robustness of Engineered Bacteria to Nutrient Stress Using Programmed Proteolysis

[Image: see text] The use of short peptide tags in synthetic genetic circuits allows for the tuning of gene expression dynamics and release of amino acid resources through targeted protein degradation. Here, we use elements of the Escherichia coli and Mesoplasma florum transfer-mRNA (tmRNA) ribosome rescue systems to compare endogenous and foreign proteolysis systems in E. coli. We characterize the performance and burden of each and show that, while both greatly shorten the half-life of a tagged protein, the endogenous system is approximately 10 times more efficient. On the basis of these results we then demonstrate using mathematical modeling and experiments how proteolysis can improve cellular robustness through targeted degradation of a reporter protein in auxotrophic strains, providing a limited secondary source of essential amino acids that help partially restore growth when nutrients become scarce. These findings provide avenues for controlling the functional lifetime of engineered cells once deployed and increasing their tolerance to fluctuations in nutrient availability

Reversible and Rapid Transfer-RNA Deactivation as a Mechanism of Translational Repression in Stress

Stress-induced changes of gene expression are crucial for survival of eukaryotic cells. Regulation at the level of translation provides the necessary plasticity for immediate changes of cellular activities and protein levels. In this study, we demonstrate that exposure to oxidative stress results in a quick repression of translation by deactivation of the aminoacyl-ends of all transfer-RNA (tRNA). An oxidative-stress activated nuclease, angiogenin, cleaves first within the conserved single-stranded 3′-CCA termini of all tRNAs, thereby blocking their use in translation. This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase]. Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs.</p

but not α-helices, β-nanotubes, β-pseudohelices, or steric zippers

A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the α-helical conformer of polyglutamine is very stable, dimers of α-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, β-nanotube, and β-pseudohelix conformers are also too short-lived to initiate aggregation. The β-hairpin- containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which β -hairpin-containing conformers act as templates for fibril formation

Global and local depletion of ternary complex limits translational elongation

The translation of genetic information according to the sequence of the mRNA template occurs with high accuracy and fidelity. Critical events in each single step of translation are selection of transfer RNA (tRNA), codon reading and tRNA-regeneration for a new cycle. We developed a model that accurately describes the dynamics of single elongation steps, thus providing a systematic insight into the sensitivity of the mRNA translation rate to dynamic environmental conditions. Alterations in the concentration of the aminoacylated tRNA can transiently stall the ribosomes during translation which results, as suggested by the model, in two outcomes: either stress-induced change in the tRNA availability triggers the premature termination of the translation and ribosomal dissociation, or extensive demand for one tRNA species results in a competition between frameshift to an aberrant open-reading frame and ribosomal drop-off. Using the bacterial Escherichia coli system, we experimentally draw parallels between these two possible mechanisms

Arts and Humanities: Senate Report (1976): Report 02

<p>(A) Translation profile of wild-type M5bG7 (top diagram, original profile) and upon introduction of translational attenuation sites (bottom diagram, adapted profile) in <i>E</i>. <i>coli</i>. Secondary structure alignment (β-sheets—blue bars, α-helices—dark red bars, uncolored empty space—linking structural elements) of M5bG7 to EH-Ar to identify regions for introduction of slow-translating stretches (indicated with arrows). The domains in M5bG7 were delineated based on the domain architecture of EH-Ar represented in the color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127039#pone.0127039.g001" target="_blank">Fig 1A</a>. Note the longer N-terminal domain of M5bG7 than that of EH-Ar. (B) Representative immunoblot of M5bG7 (abbreviated M5) variants. L1 (Leu 189, CUC/A, numbering is according to the M5bG7 sequence) and L2 (Leu 257, CUG/A) denote the synonymous exchange of a fast-translating Leu codons to Leu CUA in the first and second attenuation, respectively, and LL (Leu189, CUC/A, Leu257, CUG/A) in both simultaneously. T, total protein, S, soluble and I, insoluble fraction. GAPDH served as loading control. (C) Quantification of immunoblots of three biological replicates ± SEM. **, p<0.01, Tukey’s test. For details refer to the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127039#pone.0127039.g001" target="_blank">Fig 1</a>. (D) Quantification of mRNA levels by qRT-PCR of the M5bG7 variants. Values were normalized to GAPDH mRNA expression, represented as a fold-change to the wild-type mRNA and are means ± SEM (n = 3).</p

Trade-offs between tRNA abundance and mRNA secondary structure support smoothing of translation elongation rate

Translation of protein from mRNA is a complex multi-step process that occurs at a non-uniform rate. Variability in ribosome speed along an mRNA enables refinement of the proteome and plays a critical role in protein biogenesis. Detailed single protein studies have found both tRNA abundance and mRNA secondary structure as key modulators of translation elongation rate, but recent genome-wide ribosome profiling experiments have not observed significant influence of either on translation efficiency. Here we provide evidence that this results from an inherent trade-off between these factors. We find codons pairing to high-abundance tRNAs are preferentially used in regions of high secondary structure content, while codons read by significantly less abundant tRNAs are located in lowly structured regions. By considering long stretches of high and low mRNA secondary structure in Saccharomyces cerevisiae and Escherichia coli and comparing them to randomized-gene models and experimental expression data, we were able to distinguish clear selective pressures and increased protein expression for specific codon choices. The trade-off between secondary structure and tRNA-concentration based codon choice allows for compensation of their independent effects on translation, helping to smooth overall translational speed and reducing the chance of potentially detrimental points of excessively slow or fast ribosome movement.European Commission (Marie-Curie Actions Initial Training Network for Integrated Cellular Homeostasis (NICHE) project 289384

A Minimal Model of Ribosome Allocation Dynamics Captures Trade-offs in Expression between Endogenous and Synthetic Genes

Cells contain a finite set of resources that must be distributed across many processes to ensure survival. Among them, the largest proportion of cellular resources is dedicated to protein translation. Synthetic biology often exploits these resources to execute orthogonal genetic circuits, yet the burden this places on the cell is rarely considered. Here, we develop a minimal model of ribosome allocation dynamics capturing the demands on translation when expressing a synthetic construct together with endogenous genes required for maintenance of cell physiology. Critically, it contains three key variables related to design parameters of the synthetic construct covering: transcript abundance, translation initiation rate, and elongation time. We show that model-predicted changes in ribosome allocation closely match experimental shifts in synthetic protein expression rate and cellular growth. Intriguingly, the model is also able to accurately infer transcript levels and translation times after further exposure to additional ambient stress. Our results demonstrate that a simple model of resource allocation faithfully captures the redistribution of protein synthesis resources when faced with the burden of synthetic gene expression and environmental stress. The tractable nature of the model makes it a versatile tool for exploring the guiding principles of efficient heterologous expression and the indirect interactions that can arise between synthetic circuits and their host chassis due to competition for shared translational resources

An expanded CAG repeat in Huntingtin Causes +1 frameshifting

Maintenance of triplet decoding is crucial for the expression of functional protein because deviations either into the −1 or +1 reading frames are often non-functional. We report here that expression of huntingtin (Htt) exon 1 with expanded CAG repeats, implicated in Huntington pathology, undergoes a sporadic +1 frameshift to generate from the CAG repeat a trans-frame AGC repeat-encoded product. This +1 recoding is exclusively detected in pathological Htt variants, i.e. those with expanded repeats with more than 35 consecutive CAG codons. An atypical +1 shift site, UUC C at the 5′ end of CAG repeats, which has some resemblance to the influenza A virus shift site, triggers the +1 frameshifting and is enhanced by the increased propensity of the expanded CAG repeats to form a stem-loop structure. The +1 trans-frame-encoded product can directly influence the aggregation of the parental Htt exon 1

Polymerase III transcription is necessary for T cell priming by dendritic cells

Exposure to microbe-associated molecular patterns (MAMPs) causes dendritic cells (DCs) to undergo a remarkable activation process characterized by changes in key biochemical mechanisms. These enhance antigen processing and presentation, as well as strengthen DC capacity to stimulate naïve T cell proliferation. Here, we show that in response to the MAMPS lipopolysaccharide and polyriboinosinic:polyribocytidylic acid (Poly I:C), RNA polymerase III (Pol lII)-dependent transcription and consequently tRNA gene expression are strongly induced in DCs. This is in part caused by the phosphorylation and nuclear export of MAF1 homolog negative regulator of Poll III (MAF1), via a synergistic casein kinase 2 (CK2)- and mammalian target of rapamycin-dependent signaling cascade downstream of Toll-like receptors (TLRs). De novo tRNA expression is necessary to augment protein synthesis and compensate for tRNA degradation driven by TLR-dependent DC exposure to type-I IFN. Although protein synthesis is not strongly inhibited in absence of RNA Pol III activity, it compromises the translation of key DC mRNAs, like those coding for costimulatory molecules and proinflammatory cytokines, which instead can be stored in stress granules, as shown for CD86 mRNA. TLR-dependent CK2 stimulation and subsequent RNA Pol III activation are therefore key for the acquisition by DCs of their unique T cell immune-stimulatory functions.publishe

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as β-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translationTU Berlin, Open-Access-Mittel – 202