Mutations in the chromodomain-like insertion of translation elongation factor 3 compromise protein synthesis through reduced ATPase activity.

Translation elongation is mediated by ribosomes and multiple soluble factors, many of which are conserved across bacteria and eukaryotes. During elongation, eukaryotic elongation factor 1A (eEF1A; EF-Tu in bacteria) delivers aminoacylated-tRNA to the A-site of the ribosome, whereas eEF2 (EF-G in bacteria) translocates the ribosome along the mRNA. Fungal translation elongation is striking in its absolute requirement for a third factor, the ATPase eEF3. eEF3 binds close to the E-site of the ribosome and has been proposed to facilitate the removal of deacylated tRNA from the E-site. eEF3 has two ATP binding cassette (ABC) domains, the second of which carries a unique chromodomain-like insertion hypothesized to play a significant role in its binding to the ribosome. This model was tested in the current study using a mutational analysis of the Sac7d region of the chromodomain-like insertion. Specific mutations in this domain result in reduced growth rate as well as slower translation elongation. In vitro analysis demonstrates that these mutations do not affect the ability of eEF3 to interact with the ribosome. Kinetic analysis revealed a larger turnover number for ribosomes in comparison to eEF3, indicating that the partial reactions involving the ribosome are significantly faster than that of eEF3. Mutations in the chromodomain-like insertion severely compromise the ribosome stimulated ATPase of eEF3, strongly suggesting that it exerts an allosteric effect on the hydrolytic activity of eEF3. The chromodomain-like insertion is, therefore, vital to eEF3 function and may be targeted for developing novel antifungal drugs.

Life span extension by glucose restriction is abrogated by methionine supplementation: Cross-talk between glucose and methionine and implication of methionine as a key regulator of life span.

Caloric restriction (CR) is known to extend life span across species; however, the molecular mechanisms are not well understood. We investigate the mechanism by which glucose restriction (GR) extends yeast replicative life span, by combining ribosome profiling and RNA-seq with microfluidic-based single-cell analysis. We discovered a cross-talk between glucose sensing and the regulation of intracellular methionine: GR down-regulated the transcription and translation of methionine biosynthetic enzymes and transporters, leading to a decreased intracellular methionine concentration; external supplementation of methionine cancels the life span extension by GR. Furthermore, genetic perturbations that decrease methionine synthesis/uptake extend life span. These observations suggest that intracellular methionine mediates the life span effects of various nutrient and genetic perturbations, and that the glucose-methionine cross-talk is a general mechanism for coordinating the nutrient status and the translation/growth of a cell. Our work also implicates proteasome as a downstream effector of the life span extension by GR.

Potassium restriction boosts vacuolar acidity and extends lifespan in yeast.

Lysosome function is compromised during aging and in many disease states. Interventions that promote lysosomal activity and acidification are thus of prime interest as treatments for longevity and health. Intracellular pH can be controlled by the exchange of protons for inorganic ions, and in cells from microbes to man, when potassium is restricted in the growth medium, the cytoplasm becomes acidified. Here we use a yeast model to show that potassium limited-cells exhibit hallmarks of increased acidity in the vacuole, the analog of the lysosome, and live long by a mechanism that requires the vacuolar machinery. The emerging picture is one in which potassium restriction shores up vacuolar acidity and function, conferring health benefits early in life and extending viability into old age. Against the backdrop of well-studied protein and carbohydrate restrictions that extend lifespan and healthspan, our work establishes a novel pro-longevity paradigm of inorganic nutrient limitation.

Multiple Sclerosis-ayurvedic Management And Preventive Aspects

Multiple Sclerosis (MS) is a chronic progressive disease with a variety of cognitive, motor and sensory deficits. It is characterised by demyelination of axons of the brain and spinal cord where the patient presents with the complaints of blurred or double vision, lack of coordination, loss of balance, numbness and tremors throughout the body or extremities with weakness, muscle spasms etc. In Ayurveda, Multiple Sclerosis can be correlated to Snayusada or the aggravation of Vata in Snayu. Modern treatments like immunosuppressant, corticosteroids, stem cell therapy are highly expensive when compared to Ayurvedic management. The scope of Ayurvedic Management and preventive aspects mainly concentrates on improving the quality of life and decrease dependency of patients. The objective of this review is to evaluate the clinical utility of the Ayurvedic medicines for Multiple Sclerosis. Clinical trials by Ayurvedic experts suggest that Ayurvedic treatment processes and preventive measures (Life style management and recommended diet) may reduce the risk of Multiple Sclerosis. Here I briefly explain the current literature of Multiple Sclerosis, modern and Ayurvedic views, Ayurvedic medicines usually given in Multiple Sclerosis cases with a focus on designing prevention and treatment protocol.

The many roles of the eukaryotic elongation factor 1 complex.

The vast majority of proteins are believed to have one specific function. Throughout the course of evolution, however, some proteins have acquired additional functions to meet the demands of a complex cellular milieu. In some cases, changes in RNA or protein processing allow the cell to make the most of what is already encoded in the genome to produce slightly different forms. The eukaryotic elongation factor 1 (eEF1) complex subunits, however, have acquired such moonlighting functions without alternative forms. In this article, we discuss the canonical functions of the components of the eEF1 complex in translation elongation as well as the secondary interactions they have with other cellular factors outside of the translational apparatus. The eEF1 complex itself changes in composition as the complexity of eukaryotic organisms increases. Members of the complex are also subject to phosphorylation, a potential modulator of both canonical and non-canonical functions. Although alternative functions of the eEF1A subunit have been widely reported, recent studies are shedding light on additional functions of the eEF1B subunits. A thorough understanding of these alternate functions of eEF1 is essential for appreciating their biological relevance.

Eukaryotic polyribosome profile analysis.

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells. A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results. This procedure is readily adaptable to mammalian cells.