Mitochondrial protein synthesis assay
There are about twenty different types of amino acids; each protein is built up from a large number of them.
IN VITRO ASSAY TO DETECT INHIBITORS OF PROTEIN …
Bioinformatics 21: 3369-3376).
- The underlying assumption is that globular proteins are composed of amino acids which have the potential to form a large number of favorable interactions, whereas intrinsically unstructured proteins (IUPs) adopt no stable structure because their amino acid composition does not allow sufficient favorable interactions to form.
Here we demonstrate that EF4 does not in fact prevent misincorporations, consistent with observations in vivo (), but rather EF4 stimulates protein synthesis by up to five times in conditions of high (14 mM) Mg2+ ( A and B) or by about 50% at lower temperature (20°C) and moderate salt conditions (D, light-blue columns). This surprising observation was not anticipated considering the back-translocation function of the factor and calls for another explanation of the role of EF4 that has to be reconciled with the in vivo data presented here.
Protein synthesis rate in the cortices of BDNF mutant mice
Elongation factor 4 (EF4) is one of the most conserved proteins present in bacteria as well as in mitochondria and chloroplasts of eukaryotes. Although EF4 has the unique ability to catalyze the back-translocation reaction on posttranslocation state ribosomes, the physiological role of EF4 remains unclear. Here we demonstrate that EF4 is stored at the membrane of Escherichia coli cells and released into the cytoplasm upon conditions of high ionic strength or low temperature. Under such conditions, wild-type E. coli cells overgrow mutant cells lacking the EF4 gene within 5–10 generations. Elevated intracellular Mg2+ concentrations or low temperature retard bacterial growth and inhibit protein synthesis, probably because of formation of aberrant elongating ribosomal states. We suggest that EF4 binds to these stuck ribosomes and remobilizes them, consistent with the EF4-dependent enhancement (fivefold) in protein synthesis observed under these unfavorable conditions. The strong selective advantage conferred by the presence of EF4 at high intracellular ionic strength or low temperatures explains the ubiquitous distribution and high conservation of EF4.
Therefore we set out to study the physiological importance of the back-translocation function of EF4. Here we demonstrate that with increasing intracellular ionic strength the cellular localization of EF4 is shifted from the membrane to the cytoplasm, changing the cytoplasmmembrane ratio from 0.251 to 51. This ratio suggests that the membrane functions as a storage place for EF4, releasing the protein when needed. Accordingly, we find that the presence of the lepA gene only provides a viability advantage for the cell under unfavorable growth conditions. At high intracellular ionic strength, the fraction of unscheduled stalled ribosomes is dramatically increased and impairs synthesis rate and cotranslational folding of proteins. We find that EF4 does not prevent misincorporation, in agreement with ref. , but rather remobilizes the stalled ribosomes and thus restores the rate of protein synthesis and supports domain folding of the nascent peptide chain.
the rate of mitochondrial protein synthesis
To know whether a specific protein binds to its partner, for example, whether TFIIH interacts with TFIIE or TFIIF to complete the pre-initiation complex in transcription, different methods such as co-immunoprecipitation (co-IP), glutathione-S-transferase (GST) pull down assays, yeast-two-hybrid (Y2H) assays, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) spectros...
MoRFpred is a computational tool for sequence-based prediction and characterization of short disorder-to-order transitioning binding regions in proteins which identifies all MoRF types (a, ß, coil and complex).
The rate of protein synthesis …
Protein Synthesis -Translation and Regulation
The BCA Protein Assay Kit ..
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Fixed metabolic costs for highly variable rates of protein synthesis in sea urchin embryos and larvae
Gene Synthesis - DNA Synthesis - from .11/bp - …
SLIRP Regulates the Rate of Mitochondrial Protein Synthesis and Protects ..
Glossary | Linus Pauling Institute | Oregon State University
First, the presence of protein aggregates in tissues is hallmark of more than 40 different human disorders, from neurodegenerative diseases such as Alzheimer’s, Parkinson’s, or the transmissible spongiform encephalopathies to nonneurodegenerative systemic and localized amyloidosis, as senile systemic amyloidosis, type 2 diabetes, or even some types of cancer....
Translation (biology) - Wikipedia
The EF4-dependent stimulation of poly(Phe)-synthesis probably represents a minimal value, because the standard poly(Phe)-synthesis assay utilizes S100 fractions that already contain some EF4. To assess more precisely the stimulation factor of EF4, we analyzed the effect of EF4 in a purified system containing only EF-Tu, EF-Ts, and EF-G, together with precharged Phe-tRNA and poly(U) mRNA. In this system, we observed a maximal incorporation of about seven Phe/70S, because only 10 precharged Phe-tRNA/70S were present in the assay. Again no significant effect of EF4 was seen under optimal conditions (4.5 mM Mg2+ plus polyamines), whereas at 14 mM Mg2+ (plus polyamines) EF4 increased the rate approximately fivefold (B, Left). At 30 mM Mg2+ protein synthesis is practically blocked, yet under these extremely restricted conditions EF4 allows for slow but significant protein synthesis, indicating the mobilizing effect of EF4 on the ribosome (B, Right).
Models of protein and amino acid requirements for cattle
High concentrations (10–15 mM) of Mg2+ influence the structure of the ribosome, impairing the accuracy and reducing the rate of protein synthesis. Error-inducing aminoglycoside antibiotics such as streptomycin also impair both accuracy and rate of protein synthesis, but stimulates poly(Phe) synthesis (). However, the underlying mechanisms of error induction must be quite different in the two cases because EF4 reverses only the salt-induced effects rather than those induced by aminoglycosides (). One proposed explanation was that aminoglycosides induce a localized change of the decoding center, whereas high salt and particularly high Mg2+ is likely to cause a more general conformational change in the ribosome; these conformational changes might occasionally impair EF-G-dependent translocation in such a way that the stalled ribosomes display the codon improperly at the decoding center in the A site, thus increasing the misincorporations (). In this model, EF4 would recognize such defective translocated ribosomes and trigger a back-translocation, thereby preventing a possible misincorporation and providing a second chance for correct translocation. Substoichiometric amounts of EF4 relative to ribosomes were shown to be sufficient for increasing the active fraction. The specificity for impaired ribosomes was lost at high EF4 levels, resulting in inhibition of protein synthesis via futile cycles of translocation and back-translocation, consistent with the toxic effect of EF4 overexpression ().
Wnt Inhibition | Wnt Inhibitor Review
Under moderate conditions such as pH 7 and 37°C, EF4 is only present in trace amounts in the cytoplasm, whereas large amounts are found in the membrane fraction, as noted previously (). Because EF4 is not needed under these mild conditions, little phenotype is observed when the EF4 gene was knocked out (). In striking contrast, a dramatic shift of EF4 from the membrane to the cytoplasm is observed under specific stress conditions, for example, the EF4 cytosolmembrane distribution changes from 0.251 to 51 at high Mg2+ (). When the intracellular Mg2+ concentration is raised rapidly in response to high salt concentrations in the media, the bacterial cell might not have time enough to synthesize the 599-aa-long EF4 (indeed, E. coli EF4 is markedly longer than an average bacterial protein with 300–400 amino acids), but rather exploits the membrane as a storage vessel for EF4.
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