With the negative feedback loop now incorporated, the curve Figure 5I ; red line is much smoother, maintaining the descending order in the current almost perfectly. Therefore, the proportion of different types of proteins in the cell is maintained despite Gln4 depletion, and the inclusion of feedback restores the composition of the cell at the functional level.
Importantly however, the model incorporating feedback makes a clear, non-intuitive prediction: The charging levels of the glutamine tRNAs stay almost constant, decreasing only slightly as Gln4p is depleted Figure 5F and G. This is in contrast to the prediction made by the model without feedback.
Intuitively, this is because with feedback, the initiation rate decreases as we deplete the system of Gln4p. The charged level of glutamine tRNAs thus only decreases slightly with doxycycline treatment. Hence, the feedback mechanism homeostatically restores the balance between the rate at which glutamine tRNAs are charged and the rate at which they are used. This slight decrease in the charging level of glutamine tRNAs is enough to cause an increase in the charging level of non-glutamine tRNAs: As we have seen, the decrease in the Gln-synthetase levels causes a drop of global translation rate.
As a consequence, the demand for non-Gln tRNAs also decreases. However, the fact that the V max of non-Gln synthetases are unaffected by doxycycline leads to an increase in the charged levels of non-Gln tRNAs. Note that the feedback mechanism is only effective in the presence of doxycyline, i. In summary, the simulation results including the negative feedback loop are consistent with the lack of ribosomal queuing observed in the polysome analysis Figure 4 , as well as the impact that depletion of Gln4p has on the growth rate.
The model also makes a clear, non-intuitive prediction, namely that the charging level of the glutamine tRNAs remains approximately constant across a range of doxycycline concentrations, since a fewer number of ribosomes start translation, thereby limiting the demand for charged glutaminyl tRNAs. All other tRNAs become over-charged due to an excess charging capacity. We therefore set out to validate this prediction experimentally, below. The mathematical model makes the clear, non-intuitive prediction that the charging level of a given tRNA stays almost constant as its tRNA synthetase is depleted from the system, whereas the charging level of all the other tRNAs increases.
In the case of the lysyl-tRNA synthetase shut-off, the results clearly show that as the concentration of doxycycline increases, as the model predicts the proportion of charged lysine tRNA does not reduce, even increasing somewhat Figure 7A and B. The control, arginyl-tRNA charging levels also remained constant across a range of doxycycline concentrations and decreasing growth rates Figure 7A and B. In the case of the glutaminyl tRNA synthetase shut-off, there was also no reduction in the ratio of charged to un-charged tRNA in response to doxycycline treatment Figure 7C and D.
In parallel, the control tRNA, in this case lysyl-tRNA, also did not reduce in response to doxycycline, and gradually increased as the model predicts Figure 5E. The slower migration position of acylated tRNA is indicated closed arrowhead. Results shown are typical of other biologically independent experimental replicates. There is a linear relationship between signal and image intensity. The experimental result therefore validates the model predictions, and explains that as the activity of any individual tRNA synthetase decreases, the charging level of its target tRNA is paradoxically preserved, because the rate of translation initiation decreases as growth rate and therefore ribosome content also reduces.
The translational demand for charged glutamine tRNAs is therefore matched with the capacity of the synthetase to supply them. Moreover, there is some experimental evidence that the charging level of the other tRNAs may increase as a consequence, as predicted by the model. The model prediction that the levels of uncharged glutamine tRNA change only slightly with increasing doxycycline is validated by the tRNA Northern blot experimental observation Figures 5 and 7.
These results are however apparently at odds with the earlier observations that treating the tetO-GLN4 cells with doxycycline induces a GCN4 amino acid starvation response, indicative of the accumulation of uncharged tRNA. This suggests a hypothesis that under normal physiological conditions, some proportion of the uncharged tRNA population may be effectively sequestered by the tRNA synthetase.
Analysis of the levels of free tRNA in a synthetase-depleted strain using model simulation. Panel B ; Resulting plot of the steady state charging levels of the glutamine tRNAs as a function of the Gln4 protein ratio; using the Gillespie algorithm, the three-state-network of all 20 amino acids was simulated for different doxycycline factors. We refer to these two different states as to bound and charged. In the bound state, the tRNA is bound to the synthetase but uncharged, i.
Our model predicts how the balance between the three different tRNA states empty , bound and charged changes depending on Gln4p availability. In the presence of doxycycline, due to the reduced Gln4 synthetase concentration, the bound level lessens in favour of the empty level. During the transition between the charged and empty state, the amino acid is transferred to the nascent polypeptide.
Therefore, the transition rate between the charged and empty state is given by ribosomal current translation rate. This reflects the fact that if the growth rate is not affected by doxycycline, then the charged tRNA usage rate should not be affected either. However, as the growth rate decreases, the initiation rate also decreases, thereby lowering the charged tRNA usage rate.
The resulting synthetase sequestration model in Figure 8A simulated using a multiple-copy 3-state network; Supplementary Information considers a charging cycle for each amino acid and a total number of tRNAs in multiples of the corresponding gene copy number.
Interestingly, due to the coupling feedback factor, this relatively simple model reproduces the predicted charging level of non-glutamine tRNAs in the GTM: the charging level lysine is shown increases in the presence of doxycycline Figure 8B.
The coupling feedback factor reflects the decreasing growth rate, and translation rate of the cell in the presence of doxycycline. However, doxycycline only reduces Gln4p charging rate, therefore charging of non-glutamine amino acids then becomes the dominating form of transition in the process.
This result suggests that with the reduction in population of glutamine tRNA synthetases, although the total amount of uncharged tRNA remains more or less unchanged as shown experimentally in Figure 7 , doxycycline depletes Gln4p, causing a reduced capacity to sequester uncharged tRNA. Underscoring their importance, there is a growing list of human genetic diseases linked to human tRNA synthetase genes.
These cause a range of neurological and neurodevelopmental defects 16 , 70 , To better understand the molecular aetiology of such disorders, we established a doxycycline-dependent glutamine tRNA synthetase shut-off system in which levels of tRNA synthetase could be controlled by doxycycline in a dose-dependent manner.
Our initial characterization of synthetase depletion effects using quantitative Western blotting confirmed reductions in growth rate when the Gln4p cellular content drops below approximately 20 copies per cell, in close agreement with estimates for the physiological Gln4p abundance in yeast of 17 molecules per cell 58 , This indicates a tuned maintenance of tRNA synthetase levels in the cell sufficient to support the translational demands of the elongating ribosome population.
Intuitively, during active growth, a restriction in the charging capacity of tRNA synthetase might be expected to generate increased levels of uncharged tRNA, which activate Gcn2 kinase. Gcn2 can also be activated in a deacylated tRNA-independent manner in mouse 72 although it is not certain this happens in yeast.
This chain of events forms part of the so-called Integrated Stress Response ISR , a series of sensors and responses that focus on regulation of protein synthesis in response to a range of imposed stresses In fact, for both Gln- and Lys-tRNA synthetase shut-off events, our studies revealed just such an effect in response to doxycycline, with induction of a lacZ reporter gene under GCN4 translational control Figure 2 and Supplementary Figure S7 , and a cell-wide transcriptional response highly similar to a GCN4 transcriptional response 66 ; Figure 3.
We considered additionally that reduced levels of charged tRNA might cause mistranslation, but tRNA synthetase depletion did not cause enhanced sensitivity to the translation error-inducing drug hygromycin B Supplementary Figure S7.
Although increased levels of uncharged glutamine tRNAs should cause ribosomal queuing at CAA and CAG glutamine codons, in fact there was no evidence of shifts to larger polysomes in response to doxycycline Figure 4. However, ablating the GCN2 kinase gene during doxycycline restriction of Gln4 did not reveal any shift to larger polysomes Figure 4. In order to understand why this might be so, we applied mathematical modelling to simulate the restriction in supply of charged tRNA to translation.
The global model of translation used is to the best of our knowledge the first model that tracks the charged status of all tRNAs as they cycle through translation, become deacylated, and are then recharged by tRNA synthetases.
The model tracks a fixed population of ribosomes as they initiate, elongate and terminate on a population of mRNAs with codon compositions matching those of the GO-Slim gene ontologies. It thus provides a fine-grained view of how translation responds to changes in the cellular Gln4p levels. The model confirmed a series of experimental observations. First, simulating a reduction in the concentration of Gln4p produced slowed rates of translation indicated in the model as a reduced current of ribosomes; Figure 5D , matching experimental observation Figure 1.
Slow growth will drive slower rates of initiation, via mass action principles. This was implemented in the model by the introduction of feedback; reductions in Gln4 levels drive reduced rates of translation initiation. Simulations including the feedback effect mirrored experimental observation, namely that doxycycline restriction of Gln4 causes a global slow-down of translation initiation that prevents ribosomal queue formation Figure 5H.
Crucially however, the model made two testable predictions; first, that non-glutamine tRNA populations should become fully charged with amino acid during doxycycline treatment. Essentially, the entire translation system is regulated by the availability of Gln-tRNA Gln , and other tRNAs are utilised at slower rates, creating excess capacity in the population of 19, non-Gln synthetase species.
The second prediction was that paradoxically, there should be only minor reductions in the levels of charged glutamine tRNAs, despite our detection of a GCN4 response in the presence of doxycycline. Nevertheless, Northern blot analysis of tRNA charging confirmed both predictions Figure 7 ; the tRNA Lys population became increasingly charged in a doxycycline-responsive manner during Gln tRNA synthetase shut-off, while the levels of charged glutamine tRNA were essentially unaltered across a range of doxycycline concentrations.
The maintenance of tRNA charging, despite synthetase depletion was also seen the in case of the lysyl-tRNA in the second lysyl-tRNA synthetase shut-off strain, validating the model predictions. To address the intriguing, apparent contradiction of a GCN4 starvation response but with unaltered levels of tRNA Gln charging, a minimal synthetase sequestration model simulated the effects of reducing cellular Gln4p synthetase, while levels of charged, and uncharged tRNA were monitored.
However, in the presence of doxycycline, levels of Gln4p drop, and the ability of the Gln4p population to sequester uncharged tRNA during the charging process is diminished. This explains the clear signature of the GCN4 response in response to doxycycline while tRNA charging levels are maintained Figures 2 and 3. Our study makes clear predictions concerning the molecular aetiology of the human tRNA synthetase gene mutations which cause a wide range of neurological, and neurodevelopmental defects The model predicts potentially distinct effects on accumulation of unbound tRNA depending on whether a mutation alters the catalytic rate constant k cat of the tRNA synthetase, the affinity K M of the synthetase for tRNA or its expression level.
The latter mimics the doxycycline shut-off model implemented in our study, which triggers a GCN4 response. For example, valine synthetase VARS mutations that cause microcephaly and seizures can cause both loss of charging function, and exhibit much reduced expression Supporting the link between the ISR and neurological defects, mutations in translation initiation factor eIF2B also cause a range of neurological defects, including vanishing white matter VWM 74 , Similar processes may operate in some types of tRNA synthetase mutation, where the synthetase sequestration model indicates unbound, uncharged tRNA is available to trigger a GCN4 response.
Overall the combined experimental and model simulation analysis explains how levels of charged tRNA are maintained despite the reductions in a given tRNA synthetase, because slowed growth leads to reductions in ribosome content and translational activity; this homeostatically matches demand for charged tRNA to its supply.
However, as the synthetase sequestration model presented makes clear, depletion of Gln tRNA synthetase limits its capacity to sequester tRNA during the charging reaction. Thus, a greater proportion of the uncharged tRNA is available to react with Gcn2 kinase, triggering an amino acid starvation GCN4 response. Our study reveals fundamental new insight into the role of the tRNA synthetase population in sequestering uncharged tRNAs, thus limiting the ability of the uncharged tRNA population to interact with the Gcn2 kinase, central to triggering a GCN4 response.
The study further identifies how the biochemical properties conferred by any given tRNA synthetase mutant may confer distinct molecular and thus disease phenotypes in human. Separate from the RNA seq analysis, this research uses systems biology approaches and mathematical modelling; both models have been made publically available listed and linked in the Supplementary Material file.
The authors gratefully acknowledge the RNA sequencing and mass spectrometry support provided by the University of Aberdeen's Centre for Genome Enabled Biology and Medicine, and Proteomics respectively. IS conducted GCN4 assays.
Polysome profiling and eIF2 characterisation analysis was carried out by B. Present address: Matthew R. Warner J. The economics of ribosome biosynthesis in yeast. Trends Biochem. Google Scholar. Rodnina M. Recent mechanistic insights into eukaryotic ribosomes.
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Optimized protein extraction for quantitative proteomics of yeasts. PLoS One. Harlow E. Antibodies: A Laboratory Manual. Google Preview. Biomass-corrected supernatants were applied onto a nitrocellulose paper and blotted with an anti-mouse HRP-conjugated antibody. F Quantification of the dot blot from E. See also Figure 3—figure supplement 1. Summary data and statistics for O-propargyl-puromycin assays, GFP reporter assays and IgG secretion assay presented in Figure 3 and Figure 3—figure supplement 1.
Although the OPP incorporation assay provides a general measure of translational activity, it does not necessarily reflect a capacity of a cell to successfully produce complete, functional proteins — a property critical for maintaining cell viability, avoiding proteotoxic stress and allowing translation of stress-adaptive factors.
Due to its short half-life, d2GFP must be continuously synthesized in order to be maintained at a stable level within the cell. Indeed, a 6 hr amino acid deprivation led to a precipitous decline in d2GFP signal, while having no effect on the stable GFP levels Figure 3—figure supplement 1C. This effect could not be explained by an increase in clearance of d2GFP from amino-acid-deprived cells, as measured by a cycloheximide chase Figure 3—figure supplement 1D. Finally, CB treatment did not prevent the clearance of d2GFP from cells in which translation was arrested with cycloheximide, suggesting that CBdriven increase in d2GFP was not due to a non-specific interference of CB with the protein degradation machinery Figure 3D.
To further validate the conclusion that glutaminase inhibition augments translational capacity in amino-acid-limiting conditions, we asked whether synthesis of secreted proteins by a dedicated protein-producing cell, such as a plasma cell, which is estimated to secrete its entire protein weight in immunoglobulin per day, would be similarly bolstered by glutaminase inhibitors in amino-acid-limited conditions.
As expected, IgG yield by A20 cells was reduced when amino acids were limiting, yet a concomitant inhibition of glutaminase with CB restored it to a level comparable to that seen in amino-acid-replete cells Figure 3E,F. Taken together, these results indicate that the selective loss of charged tRNA Gln limits translation in amino-acid-depleted cells, which can be counteracted by adding exogenous glutamine or blocking glutamine consumption in a glutaminolysis reaction.
Glutamine accounts for approximately 4. However, there are 68 proteins in the human proteome that contain uninterrupted tracts of 10 or more glutamine residues, known as polyglutamine, or polyQ, tracts. Since amino acid depletion triggers a profound depletion of charged tRNA Gln pools, we hypothesized that translation of polyQ tracts may be particularly sensitive to amino acid depletion, which would lead to a collective downregulation of polyQ-containing proteins. Furthermore, supplementing amino-acid-depleted medium with either CB or glutamine restored the levels of all four polyQ-containing proteins tested, suggesting that levels of polyQ proteins can be modulated by changes in the availability of glutamine for translation.
A MiaPaCa2 cells were treated as shown for 48 hr. Levels of indicated proteins were examined by western blotting. Levels of indicated polyQ proteins were concurrently measured by western blot. Dotted line represents the relative value of 5-EU incorporation in cells in which transcription was arrested via actinomycin D ActD pretreatment for 10 min prior to adding 5-EU.
C MiaPaCa2 cells were treated with media containing reduced quantities of glutamine or leucine or with complete medium for 48 hr, at which point new protein synthesis was assayed by puromycin incorporation. Levels of indicated polyQ and non-polyQ proteins were determined by western blotting in a parallel set of identically treated samples. Cells were treated as indicated for 48 hr, and levels of recombinant and endogenous TBP were determined by western blot.
An asterisk indicates endogenous TBP. Cells were treated with complete or amino-acid-free DMEM in presence of doxycycline for 6 hr. See also Figure 4—figure supplement 1. Summary data and statistics for 5-ethynyl-uridine assays and GFP reporter assays in Figure 4 and Figure 4—figure supplement 1.
Next, we set out to determine the minimal dose of extracellular glutamine under which cells are still able to maintain the expression of polyQ proteins. This observation indicates that glutamine concentrations found in nutrient-poor regions within solid tumors in vivo could indeed be sufficient to deplete polyQ proteins and that the regional differences in glutamine availability may establish a pattern of heterogeneous polyQ protein expression within tumors.
Indeed, we found that exposing cells to limiting amino acid conditions markedly suppressed incorporation of labeled uridine into nascent RNA, which could be partially restored by CB or added glutamine, thus mirroring the effect these conditions had on polyQ protein levels Figure 4B. To further verify the on-target nature of CBmediated restoration of polyQ protein expression and labeled uridine incorporation, we suppressed GLS expression in cells by RNA interference.
Similarly to the effects observed with CB and with exogenous glutamine supplementation, shRNA constructs targeting human GLS, but not control shRNAs, resulted in a significant rescue of both polyQ protein expression Figure 4—figure supplement 1D as well as of labeled uridine incorporation Figure 4—figure supplement 1E in amino-acid-depleted cells. Importantly, glutaminase inhibition did not increase the size of nucleotide triphosphate pools in amino-acid-depleted cells, indicating that its effect on RNA synthesis is unlikely to be explained by increased nucleotide availability under these conditions Figure 4—figure supplement 1F.
Taken together, these observations raise the possibility that the presence of polyQ tracts within transcriptional regulators may represent an amino-acid-sensing adaptation that allows a cell to modulate transcriptional output in response to changes in amino acid availability. To test this hypothesis further, we asked whether depletion of glutamine alone, but not of an unrelated amino acid, could trigger a decline in polyQ protein levels. Even though both glutamine-poor and leucine-poor conditions equivalently inhibited bulk translation, only glutamine-poor formulations led to a significant depletion of polyQ proteins Figure 4C.
Taken together, these observations indicate that depletion of glutamine specifically, and not the inhibition of translation associated with the depletion of a different amino acid, is responsible for the observed decline in polyQ protein levels. As expected, recombinant TBP with an intact polyQ tract behaved similarly to endogenous TBP protein — that is, its levels declined precipitously in amino-acid-depleted conditions and were fully restored by CB Figure 4D.
Taken together, these data indicate that the polyQ tract is required for the sensitivity of TBP to amino acid depletion. Conversely, we asked whether adding a polyQ tract to a non-polyQ protein is sufficient to make it sensitive to amino acid limitation.
Reporter expression was induced with doxycycline in presence or absence of extracellular amino acids and cells were monitored for the accumulation of GFP signal by FACS. In contrast to control GFP-expressing cells, in which the amount of fluorescence accumulation over the course of 6 hr was not affected by the absence of extracellular amino acids, PolyQ-GFP-expressing cells accumulated markedly less fluorescence in amino-acid-depleted than in complete medium Figure 4F.
This effect could not be attributed to the decreased half-life of PolyQ-GFP protein in amino-acid-deprived cells Figure 4—figure supplement 1G. Importantly, both CB and supplementing additional glutamine facilitated PolyQ-GFP accumulation in amino-acid-deprived cells, providing additional evidence that restoring charged tRNA Gln pools may ameliorate the defect in polyQ protein synthesis in an amino-acid-limited state Figure 4G.
Based on these findings, we hypothesized that amino acid deprivation may similarly reduce translational fidelity on polyQ tracts as a consequence of an imbalance in charged tRNA Gln pools relative to the rest of the tRNA compartment. A representative result out of at least three independent experiments is shown.
See also Figure 5—figure supplement 1. Summary data and statistics for GFP reporter assays presented in Figure 5 and Figure 5—figure supplement 1. This effect was completely blocked by CB Figure 5B. Notably, the extent of GFP accumulation correlated with the extent of amino acid depletion. Taken together, these observations indicate that across a variety of cellular contexts, amino acid depletion renders translation of polyglutamine-tract-containing transcripts prone to frame shifting.
In addition, we found that inhibiting glutaminase or supplying exogenous glutamine counters the loss of translational fidelity in this reporter context, which implicates the deficit of the charged form of tRNA Gln in this phenomenon. We wondered if the frame shifting effect is strictly an amino acid depletion-associated phenomenon, or if it can be triggered by other stresses as well. Only amino acid depletion-associated stress resulted in GFP accumulation, indicating that amino acid deficit is a specific trigger for translational frame shifting.
The effect of leucine depletion on frame shifting observed with both reporters is consistent with a nearly identical leucine content of PolyQ and Luc fragments 6 and 7 amino acid residues, respectively. Taken together, these observations indicate that the presence of a polyQ tract promotes translational frame shifting in response to amino acid depletion. Furthermore, our findings indicate that polyQ-associated frame shifting is triggered specifically by the depletion of glutamine rather than that of any given amino acid.
Finally, we asked whether the loss of translational fidelity triggered by amino acid depletion can be reversed by re-feeding the amino-acid-depleted cells with amino-acid-rich medium. Taken together, our observations indicate that in diverse cellular contexts, translation of polyglutamine-tract-containing transcripts is prone to frame shifting in response to amino acid deficit but not to other types of cellular stresses, and can be augmented by treatments that restore tRNA Gln to its charged state.
After 3 weeks, xenografts were harvested and paraffin-embedded tumor sections were stained with anti-GFP antibodies. Taken together, our results indicate that the translational frame shifting of polyglutamine tracts can be observed in discrete areas within solid tumors in vivo. Paraffin-embedded samples were stained with anti-GFP antibody. Representative images out of two independent experiments are shown. Relative abundance of GFP-positive cells was determined by flow cytometry.
The present work demonstrates that cells have the capacity to maintain pools of charged tRNAs during interruptions in extracellular amino acid supply. Such an ability allows the cells to retain their adaptive translational capacity for extended periods of time even when the vascular supply of free amino acids is compromised. The ability to maintain a charged tRNA compartment in amino-acid-deprived cells requires lysosomal function, which is consistent with the established role of the lysosome as a critical source of amino acids in a nutrient-limited state.
Our work further reveals that adaptive translation over time results in a selective depletion of tRNA Gln charging that reduces the net translation. This reduction in translation does not occur because cells cannot either recycle glutamine from lysosome-degraded proteins or take up sufficient extracellular glutamine when the vascular delivery of amino acids is compromised.
Instead, it results from the propensity of cells to convert glutamine to glutamate through glutaminase. Indeed, we found that inhibiting glutaminase in the context of amino acid deficit augments the capacity of amino-acid-depleted cells to maintain tRNA Gln charging and sustain protein synthesis. This indicates that even though glutaminase inhibition suppresses growth when amino acids are abundant, it facilitates translation and even cell proliferation when amino acid supply is limited.
This is suggested to be due to the use of glutamate for glutathione production Lora et al. Thus, allosteric inhibitors of glutaminase may have contrasting effects on cells residing in abundant vs. Amino acid limitation-associated depletion of tRNA Gln also results in a collective depletion of a number of key core transcription factors whose protein sequences contain polyglutamine tracts and consequently reduces cellular transcriptional output. Interestingly, a comparative sequence analysis across a broad spectrum of metazoan proteomes has revealed multiple instances in which the position of a polyQ tract is not tethered to a fixed position within the primary protein sequence among orthologs from diverse taxa.
This observation suggests that the mere presence — rather than a specific location — of a polyQ tract may be of functional importance Schaefer et al. Altogether, our observations raise the possibility that polyglutamine tracts in proteins may carry out a nutrient-sensing function by changing the protein level in response to extracellular amino acid availability. Finally, our work demonstrates that amino acid depletion is associated with the loss of translational fidelity among polyglutamine-containing transcripts, which, as we demonstrate, takes place in discrete areas of solid tumors in vivo.
This, in turn, warrants further investigation of this phenomenon as a potential tool to identify and characterize amino-acid-poor cell populations from a variety of pathophysiological contexts.
James Hsieh. James Hodge. To induce quiescence, cells were cultured for 3 days post-confluence, with culture medium changed daily. For cell proliferation experiments, cell numbers at the start and the end of the experiment were counted in triplicates using the Multisizer 3 Coulter Counter Beckman.
For amino acid deprivation experiments in adherent cells, cells were rinsed with PBS, and treatment media lacking amino acids or with all amino acids present at an indicated fraction of that in a standard formulation, was added. All chemical inhibitors were resuspended in DMSO.
An equivalent amount of DMSO was added to control samples to control for any solvent-based effects. Equal amounts of total protein were separated on NuPAGE Bis-Tris or Tris-Acetate for large proteins gels Life Technologies , transferred to nitrocellulose membranes and subjected to Western blotting with indicated primary antibodies.
Cells were treated as indicated. Lysates were shaken with chloroform , centrifuged at 18, g and precipitated with 2. Samples were resuspended in 0. Sodium periodate was from Sigma-Aldrich Reactions were quenched with glucose for 15 min.
Ct values obtained with primers specific for yeast tRNA Phe primers were subtracted from Ct values obtained with primers specific for an isodecoder of interest. The charged fraction value was calculated from a relative difference between a delta-Ct value from a non-oxidized representing total and oxidized representing charged samples for each primer pair. See also Supplementary file 1 for a full list of oligonucleotides used. Ligation products were subjected to eight cycles of PCR with primers containing 8-mer barcodes and p5 and p7 Illumina adaptors.
Reads were normalized by library size and the yeast phenylalanine tRNA spike-in counts prior to determining charge ratios.
The protocol was adapted from Jester, with some modifications. Cells were treated as described before. Sample loading was normalized to cell biomass.
Membranes were washed with 0. Membranes were stripped with boiling 0. Scott Lowe. Cheryl Arrowsmith [ Harding et al. Retroviral particles were produced by cotransfecting the viral backbone plasmid together with packaging plasmids into T cells with polyethylenimine —1, Polysciences. Starting biomass was recorded by determining starting cell volume via Coulter Counter and multiplying it by starting cell number. There are 64 possible codons arising from a combination of four nucleotides.
All tRNAs have two functions: to be chemically linked to a particular amino acid and to base-pair with a codon in mRNA so that the amino acid can be added to a growing peptide chain. Transfer RNA tRNA , small molecule in cells that carries amino acids to organelles called ribosomes, where they are linked into proteins. Ribosomes consist of two major components: the small and large ribosomal subunits.
The ribosomes and associated molecules are also known as the translational apparatus. Uracil is a nucleotide, much like adenine, guanine, thymine, and cytosine, which are the building blocks of DNA, except uracil replaces thymine in RNA. So uracil is the nucleotide that is found almost exclusively in RNA. Therefore, in prokaryotic cells, the control of gene expression is mostly at the transcriptional level. Eukaryotic cells, in contrast, have intracellular organelles that add to their complexity.
Begin typing your search term above and press enter to search. Press ESC to cancel. Ben Davis December 20, How does charging of tRNA takes place? What is the charging of tRNA? What is the difference between charged and uncharged tRNA?
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