scholarly journals Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation

2021 ◽  
Author(s):  
Simon Diez ◽  
Molly Hydorn ◽  
Abigail Whalen ◽  
Jonathan Dworkin
Keyword(s):  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Nutrient Limitation ◽  
Dynamic Environments ◽  
Phenotypic Heterogeneity ◽  
Cellular Heterogeneity ◽  
Microbial Populations ◽  
Low Protein ◽  
Synthesis Activity

Phenotypic heterogeneity of microbial populations can facilitate survival in dynamic environments by generating sub-populations of cells that may have differential fitness in a future environment. Bacillus subtilis cultures experiencing nutrient limitation contain distinct sub-populations of cells exhibiting either comparatively high or low protein synthesis activity. This heterogeneity requires the production of phosphorylated guanosine nucleotides (pp)ppGpp by three synthases: SasA, SasB, and RelA. Here we show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population of cells exhibiting low protein synthesis whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. The RelA product (pppGpp) allosterically activates SasB and we find, in contrast, that the SasA product (pGpp) competitively inhibits this activation. Finally, we provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This work therefore identifies the mechanism underlying phenotypic heterogeneity in the central physiological process of protein synthesis.

scholarly journals The alarmones (p)ppGpp directly regulate translation initiation during entry into quiescence

2020 ◽  
Vol 117 (27) ◽  
pp. 15565-15572 ◽  
Author(s):  
Simon Diez ◽  
Jaewook Ryu ◽  
Kelvin Caban ◽  
Ruben L. Gonzalez ◽  
Jonathan Dworkin
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Direct Interaction ◽  
Extended Period ◽  
Initiation Factor ◽  
Synthetic Activity ◽  
Inhibit Protein Synthesis ◽  
Low Protein ◽  
Initiation Factor 2

Many bacteria exist in a state of metabolic quiescence where energy consumption must be minimized so as to maximize available resources over a potentially extended period of time. As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for down-regulation during the transition from growth to quiescence. We observe that whenBacillus subtilisexits rapid growth, a subpopulation of cells emerges with very low protein synthetic activity. This phenotypic heterogeneity requires the production of the nucleotides (p)ppGpp, which we show are sufficient to inhibit protein synthesis in vivo. We then show that one of these molecules, ppGpp, inhibits protein synthesis by preventing the allosteric activation of the essential GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (p)ppGpp and IF2.

scholarly journals Proteostasis and Myeloid Stem Cell Production

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-42-SCI-42
Author(s):  
Robert Signer
Keyword(s):  
Stem Cells ◽  
Protein Synthesis ◽  
Stem Cell ◽  
Blood Cells ◽  
Cell Function ◽  
Trna Synthetase ◽  
Unfolded Proteins ◽  
Self Renewal ◽  
Low Protein

Hematopoietic stem cells (HSCs) regenerate blood cells lost to turnover, injury and disease. Defects in HSC maintenance, such as those that occur during aging, lead to anemia, impaired immunity, and bone marrow failure. Over- or ectopic activation of HSC self-renewal programs leads to hematopoietic neoplasms. Thus, defects in HSC maintenance can lead to diverse malignant and non-malignant hematopoietic disorders. We recently discovered that HSCs have lower rates of protein synthesis than other blood cells. Low protein synthesis is necessary for HSCs, as genetic changes that increase protein synthesis impair HSC function. Importantly, this does not simply reflect HSC quiescence, as dividing HSCs also have lower rates of protein synthesis as compared to dividing restricted progenitors. However, why stem cells depend on low protein synthesis and how increases in protein synthesis impair stem cell function remain largely unknown. Translation is a key cog in both the gene expression and protein homeostasis (proteostasis) networks, and thus influences both the content and the quality of the proteome. We have now determined that low protein synthesis within HSCs is associated with elevated proteome quality in vivo. HSCs contain less ubiquitylated and unfolded proteins as compared to restricted myeloid progenitors, and modest increases in protein synthesis cause an accumulation of misfolded/unfolded proteins within HSCs. Thus, HSCs depend upon low protein synthesis to maintain proteome quality. To test how translational control of proteome quality affects stem cell function, we examined Aarssti/sti mice that harbor a mutation in the alanyl-tRNA synthetase, which causes a tRNA editing defect that increases amino acid misincorporation errors during translation. Aarssti/sti mice exhibit reduced HSC numbers and significantly diminished serial reconstituting activity in vivo, but do not exhibit defects within restricted progenitors. Surprisingly, a modest accumulation of misfolded/unfolded proteins does not induce significant activation of the unfolded protein response within HSCs, but instead overwhelms the capacity of the proteasome, which promotes the stabilization and increased abundance of c-Myc. Conditional deletion of a single copy of Myc is sufficient to significantly rescue serial reconstitution defects in Aarssti/sti HSCs. HSCs are thus dependent on low protein synthesis to maintain proteome quality and homeostasis to preserve their self-renewal activity in vivo. Disclosures No relevant conflicts of interest to declare.

Phenotypic heterogeneity of microbial populations under nutrient limitation

2020 ◽  
Vol 62 ◽  
pp. 160-167 ◽  
Author(s):  
Ana Gasperotti ◽  
Sophie Brameyer ◽  
Florian Fabiani ◽  
Kirsten Jung
Keyword(s):  
Nutrient Limitation ◽  
Phenotypic Heterogeneity ◽  
Microbial Populations

Respiratory energy requirements and rate of protein turnover in vivo determined by the use of an inhibitor of protein synthesis and a probe to assess its effect

1994 ◽  
Vol 92 (4) ◽  
pp. 585-594 ◽  
Author(s):  
T. J. Bouma ◽  
R. De Visser ◽  
J. H. J. A. Janssen ◽  
M. J. De Kock ◽  
P H. Van Leeuwen ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Energy Requirements ◽  
Inhibitor Of Protein Synthesis

Rapid in vivo induction of leukocyte tissue factor mRNA and protein synthesis following low dose endotoxin administration to rabbits

2001 ◽  
Vol 2 (3) ◽  
pp. 188-195 ◽  
Author(s):  
Tara C Brutzki ◽  
Myron J Kulczycky ◽  
Leslie Bardossy ◽  
Bryan J Clarke ◽  
Morris A Blajchman
Keyword(s):  
Protein Synthesis ◽  
Tissue Factor ◽  
Low Dose ◽  
Endotoxin Administration ◽  
In Vivo Induction

scholarly journals LOCALIZED MUTAGENESIS FOR THE ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI AFFECTED IN PROTEIN SYNTHESIS

Genetics ◽  
1979 ◽  
Vol 91 (2) ◽  
pp. 215-227
Author(s):  
W Scott Champney
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Temperature Sensitive ◽  
Chromosomal Locus ◽  
Ribosomal Subunits ◽  
Prolonged Incubation ◽  
Temperature Sensitive Mutants ◽  
Inhibition Of Growth ◽  
Localized Mutagenesis

ABSTRACT Two variations of the method of localized mutagenesis were used to introduce mutations into the 72 min region of the Escherichia coli chromosome. Twenty temperature-sensitive mutants, with linkage to markers in this region, have been examined. Each strain showed an inhibition of growth in liquid medium at 44°, and 19 of the mutants lost viability upon prolonged incubation at this temperature. A reduction in the rate of in vivo RNA and protein synthesis was observed for each mutant at 44°, relative to a control strain. Eleven of the mutants were altered in growth sensitivity or resistance to one or more of three ribosomal antibiotics. The incomplete assembly of ribosomal subunits was detected in nine strains grown at 44°. The characteristics of these mutants suggest that many of them are altered in genes for translational or transcriptional components, consistent with the clustering of these genes at this chromosomal locus.

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