The process of protein synthesis provides cells with building blocks and regulatory Protein synthesis proceeds through three phases: initiation of mRNA . Protein Synthesis: What Is It? • During transcription a molecule of messenger. RNA (mRNA) is synthesized according to instructions provided by the DNA. Lecture Protein Synthesis. Key learning goals: • Be able to explain the main stuctural features of ribosomes, and know (roughly) how many DNA and protein.
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The Mechanism of Protein Synthesis. Problems: 2,3,6,7,9,13,14,15,18,19, • Initiation: Locating the start codon. • Elongation: Reading the codons (5'→3') and. RNA Polymerase consists of a core enzyme and a σ (sigma factor). The core enzyme consists of four peptide chains, α, β, β, ώ. • The enzyme itself is ineffective. Protein synthesis is the process in which a cell makes protein based on the message contained within its DNA. • However: – DNA is only found in the nucleus.
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The physiologic importance lies in the potential ability of amino acids as specific nutrients designed to counteract the high host protein wasting associated with a number of disease entities, including cancer, HIV infection, sepsis, and diabetes, thereby preventing potential complications and limiting debilitating morbidities.
Ultimately, mortality in septic patients is reduced 3. Likewise, BCAA-enriched oral supplementation improves morbidity and quality of life in patients undergoing major liver resection and chemo-embolization for hepatic carcinoma 6. Overview of synthesis of proteins in mammalian systems Synthesis of proteins in mammalian cells is a complex process 7 , 8 Fig.
The accelerated rates of protein synthesis appear independent of hyperacidemia. Hence, the nutrient-induced increases most likely involve processes other than amino acid availability, i.
Indeed, nutrients appear to stimulate protein synthesis largely by accelerating mRNA translation initiation faster than elongation.
Two major steps in mRNA translation initiation control protein synthesis. Oral feeding does not enhance the formation of 43S preinitiation complex 10 , The next regulatory step involves the recognition, unwinding, and binding of mRNA to the 43S preinitiation complex, catalyzed by a multi-subunit complex of eukaryotic factors referred to as eIF4F 12 — Acute provision of nutrients enhances this step, the assembly of active eIF4F complex 2 , To our knowledge, we were the first to report a positive linear relationship between rates of protein synthesis and amount of eIF4G associated with eIF4E in vivo 20 , consistent with the proposed role of eIF4G-eIF4E complex in the overall regulation of protein synthesis.
FIGURE 3 Dual function of leucine as oxidative fuel and activator of signaling pathways by direct and indirect processes adapted from FIGURE 3 View large Download slide Dual function of leucine as oxidative fuel and activator of signaling pathways by direct and indirect processes adapted from Increased phosphorylation of eIF4G correlates with accelerated rates of protein synthesis in cell extracts 32 and in striated muscle in vivo 33 — Meal feeding augmented the phosphorylation of 4E-BP1 and S6K1 Thr , both of which are associated with activation of these modulators of translation initiation.
Phosphorylation PKB, an upstream kinase responsible for phosphorylating mTOR, on Thr was only elevated after one-half hour of meal feeding, whereas phosphorylation Ser was significantly elevated up to 1 h after initiation of feeding. In cardiac muscle, a slightly different response to meal feeding was observed Leucine can substitute for a complete protein meal Of the nutrients provided by a complete meal, postprandial increases in the plasma concentration of amino acids after a mainly protein-containing meal may provide a signal for accelerating protein synthesis 37 , Infusing amino acids in vivo enhances muscle protein synthesis independent of any stimulatory effect induced by changes in plasma anabolic hormone concentrations 1 , 39 — Indeed, an oral gavage with leucine resulted in a stimulation of protein synthesis that was independent of changes of plasma insulin concentrations, whereas a gavage containing carbohydrates glucose plus sucrose that raised insulin concentrations over 2.
This does not mean that anabolic hormones are not required for the effects of amino acids on mRNA translation initiation. Indeed, administration of anti-insulin antibodies blunts the effects of refeeding to stimulate protein synthesis 45 , Moreover, when insulin secretion is retarded by somatostatin infusions 47 , oral leucine gavage was ineffective in augmenting rates of protein synthesis in skeletal muscle Amino acids probably stimulate key regulatory protein factors controlling protein synthesis rather than simply increasing substrate supply, which would be expected to enhance only peptide-chain elongation and not translation initiation 49 , Amino acids, BCAA leucine, isoleucine, and valine in particular, not only provide substrate for protein synthesis but are also now recognized to stimulate a number of cell-signaling pathways important in the regulation of mRNA translation 38 , 51 — Not all amino acids have the same potency in stimulating protein synthesis.
Leucine seems to be the most potent of the BCAA with regard to most of these effects and therefore may be the most physiologically relevant. Oral gavage with a solution containing leucine, but not isoleucine or valine, stimulates protein synthesis with an increased assembly of eIF4G, S6K1 phosphorylation, and eIF4G phosphorylation in gastrocnemius 10 , 58 — 60 and heart Similar to meal feeding, the mTOR signaling pathway has been proposed as 1 potential intracellular signaling target for mediating the effects of amino acids on mRNA translation initiation.
In adipocytes, the efficacy of amino acids in activating mTOR signaling appears to be related to their structural similarity to leucine. Thus, leucine and norleucine are posited to be agonists at a common leucine recognition site in adipocytes, LeuRa 27 , Leucine and its analog, norleucine, constitute a direct-acting nutrient signal 1 , 27 , 62 that communicates the presence of an ingested protein-containing meal to peripheral organs, including striated muscle 1 , 50 , Leucine is ideally suited for its role as a nutrient signal for several reasons.
First, plasma leucine concentrations rise rapidly after a meal and remain elevated during feeding in meal-trained rats.
Second, rises in circulating plasma leucine concentration observed after a meal are within the range over which leucine stimulates mTOR activity 27 , 62 , Third, leucine is the most efficacious amino acid in stimulating protein synthesis in skeletal muscle 38 , 65 and, more importantly, mTOR 27 , 62 , Fourth, leucine is derived from the diet and cannot be synthesized by mammals, allowing for a defined administration.
Lastly, skeletal muscle has a mechanism to regulate and eliminate this putative nutrient signal through oxidation of leucine, although this pathway is generally repressed except when plasma leucine concentrations rise Leucine stimulates mTOR phosphorylation in skeletal muscle Leucine alone can substitute for a meal in stimulating signal transduction pathways, leading to a stimulation of protein synthesis by accelerating mRNA translation initiation.
Oral leucine gavage increased muscle protein synthesis within 20 min 10 , In contrast, the m1A9 on the D arm required for the formation of the cloverleaf structure of tRNALys 25 and catalyzed by a different methyltransferase complex 26 , was detected in both wild-type and MERRF cells.
Our sequencing approach expanded the landscape of MERRF pathogenesis by implicating a novel methyl modification on tRNALys, highlighting the versatility and robustness of DM-tRNA-seq for investigating the regulation of mitochondrial gene expression disorders.
From each of the libraries, 22—50 million reads were mapped. This finding is entirely consistent with our data from proliferating myoblasts, strongly suggesting a key role of this modification in the pathogenesis of MERRF. Whereas another study showed how structurally abnormal mitochondrial tRNAs can be polyadenylated for degradation For this analysis, we sorted sequence reads into two pools based upon the presence or absence of the CCA tail.
Aminoacylation of tRNAs requires the addition of the CCA extension, which is catalyzed by a mitochondrially targeted TRNT1, and is critical for mitochondrial protein synthesis and human health 28 , These data indicate that adenylation of mitochondrial tRNAs is a far more complex process than previously thought and may play a role in the regulation of mitochondrial tRNA populations and the pathogenesis of human disease.
Importantly, TRMT61B overexpression increased the synthesis of selected proteins and suppressed the generation of aberrantly sized polypeptides Fig. The magnitude of this effect did not correlate with the number of lysine codons of a given polypeptide.
Aberrantly sized labeled polypeptides are indicated. Representative data of multiple independent experiments Full size image Next, we asked whether the increased protein synthesis with TRMT61B overexpression also modulated the short term and long-term stability of mitochondrial nascent chains.
To test if mitochondrial ribosome stability was affected by TRMT61B overexpression, we assessed the steady-state levels of mitochondrial ribosomal proteins from the small and large subunit by immunoblotting because their abundance is a reliable proxy for stability 33 but found no difference Fig.
Our data showed that restoration of the m1A58 on tRNALys can enhance translation in MERRF, arguing against this methyl modification as being a strictly negative regulator of mitochondrial gene expression as has been proposed for the m1A modification on mitochondrial mRNAs 31 , Rather, there appears to be a tight regulation of this methyl modification on mitochondrial RNAs to finely tune mitochondrial protein synthesis.
Because reduced modification of the anticodon wobble base position is implicated in MERRF pathogenesis, we wanted to test whether overexpression of the key enzymes could also modulate mitochondrial translation. MTU1 catalyzes the thiol modification of the anticodon wobble base, a reaction that is required for human health 34 , but appears to have no effect on mitochondrial protein synthesis in human fibroblasts In contrast, overexpression of MTO1 had a marked effect on mitochondrial protein synthesis and protein stability Fig.
MTO1 overexpression restored mitochondrial protein synthesis to wild-type levels Fig. Moreover, these effects were independent of the m1A58 modification Fig.
No adverse effects were observed on wild-type myoblasts overexpressing MTO1. Collectively, these findings demonstrate that post-transcriptional modifications of mitochondrial tRNAs exert differential effects on protein synthesis and stability, revealing the profound influence that RNA modifications have on mitochondrial gene expression and the pathogenesis of human disease.
Discussion RNA modifications have emerged as major regulators of gene expression across all domains of life In this study, we showed that post-transcriptional modifications on a single mitochondrial tRNA modulate nascent chain synthesis and protein stability.
Our data also highlight the importance of using diploid patient cells to investigate the regulation of mitochondrial gene expression in the pathogenesis of mitochondrial diseases. Therefore, weakened binding of the m.
Consistent with this interpretation, overexpression of the m1A58 methyltransferase, TRMT61B, restored the modification, increasing the synthesis of mitochondrial nascent chains and preventing the generation of aberrantly sized polypeptides.
Together, these data argue that the m1A58 modification is a positive regulator for mitochondrial translation elongation. Despite rescuing the translation defect, however, most nascent chains were still unstable.
These data point to a more complex molecular pathogenesis of the m. Absence of the post-transcriptional modification of the wobble anticodon position of the cytoplasmic tRNALys has been shown to cause ribosome pausing on lysine codons, inducing a severe delay in the rate of translation elongation 10 , 11 , The data from other translation systems suggest a uniform rate of decoding for all tRNAs The main differences appear to be in the stability of the codon recognition complex among tRNAs, which specifically affect the geometry of the codon—anticodon complex and disassociation rates Consistent with this interpretation, overexpressing MTO1 dramatically increased the rate of mitochondrial protein synthesis, but most of the nascent chains were still unstable, suggesting the fidelity of mitochondrial protein synthesis is still disrupted.
Our data suggest the m. Together, our data point to two post-transcriptional modifications on the tRNALys that are important for the efficiency and fidelity of mitochondrial protein synthesis. Since the 13 mitochondrial proteins are co-translationally inserted into the lipid bilayer to assemble into large heterooligomeric complexes 39 , nascent chain misfolding would interfere with the kinetics of assembly for the oxidative phosphorylation complexes.
The methyltransferase TRMT10C, which is responsible for the m1A9 modification in tRNAs, can also post-transcriptionally modify mitochondrial mRNAs, in particular MT-ND5, in a process that is developmentally regulated during embryogenesis and post-natally between germline and somatic tissues These results suggest that m1A modifications catalyzed by the same methyltransferase could have opposing effects on mitochondrial protein synthesis: an inhibitory role on mRNAs and positive role with mitochondrial tRNA.
Such a mechanism on mitochondrial mRNAs might be a part of a quality control step to ensure aberrant mRNAs are not fully engaged in translation to minimize the potential for misfolded proteins.
An elevated m1A58 level on tRNALys may compete with decoding of other tRNAs at near-cognate codons, thereby decreasing translational fidelity and ultimately promoting protein misfolding and degradation. The expression level of these enzymes could vary across different cell types and tissues, and be a key regulator for modulating mitochondrial protein synthesis. In the cytosol, dynamic regulation of the m1A modification on tRNAs in response to metabolic cues is a significant regulator of cellular protein synthesis Therefore, it is intriguing to speculate that a similar metabolic regulation of this post-transcriptional modification exists for mitochondrial gene expression.
An interesting finding of our study points to different pools of tRNAs within the organelle. This effect is exemplified by the oligoadenylation of molecules lacking the CCA extension, which implies the existence of separate pools of tRNAs with and without this post-transcriptional polyA extension.Do you know that nucleic acids and proteins are highly important to cellular function?
Use the chart below to determine the correct codons. Thus, it appears that signaling through mTOR is important for mediating the effects of leucine feeding on translation in muscle.
Finally, rapamycin reversed the leucine-dependent decrease in eIF4E phosphorylation, resulting in values that were not different from those of food-deprived rats. Diagram showing the process of translation Diagram showing the translation of mRNA and the synthesis of proteins by a ribosome Phenomena of amino acid assembly from RNA.
Second, phosphorylation of Thr causes dissociation of the tuberin-hamartin complex 81 and limits the tuberin-hamartin complex reductions of Rheb-GTPase activity, thereby relieving inhibition mTOR. Taken together, our data suggest differential regulation of the mitochondrial genome in proliferating human cultured cells, highlighting the need to use diploid cells to investigate the pathogenic mechanisms of human mitochondrial gene expression disorders.
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