BIO 344 1st Edition Lecture 17Outline of Last Lecture I. Translation: RibosomesII. Finding the Start sitea. Shine delgarnob. 5’ capc. Ternary complexd. Effect of 5’ cap on translatione. Effect of 3’ poly A tail on translationf. PolioIII. Elongationa. Ef-Tu/GTPb. Ef-Ts/GDPIV. Fidelitya. Rejecting tRNAV. TranslocationOutline of Current Lecture I. Translational controla. Amino acid starvationII. Global translation down regulated in amino acid starvationa. yeasti. Gcn2 kinase and eIF2 on Gcn4ii. Ternary complex on site selectionb. Mammalsi. HRI, PKR, PERK, Gcn2ii. Mice food selectioniii. Alcohol derivativesCurrent LectureTranslational Control- Amino acid control—amino acid starvationo Mice look for another food sourceo Yeast synthesize their own amino acidsThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute. Starvation induces a slowdown in translation but Gcn4 mRNA increases Gcn4 codes for a transcription factor that control regulation of biosynthetic genesGlobal Translation Down Regulation- Gcn2 kinase senses uncharged tRNA (low amino acids)- Gcn2 phosphorylates eIF2, which blocks its GTP exchange cycle, decreasing levels of the ternary complex, decreasing global translation- Decreased levels of ternary complex promotes use of another start site- eIF2- GTPase is activated by recognition of start codon to hydrolyze and leave as eIf2- GDPo eIF2-GTP is regenerated by GEF eIF2B- Gcn2 kinase built form several protein moduleso Kinase domain phosphorylates eIF2-alpha Adds a phosphate group (negative charge) to serineo HisRS looks like histidyl tRNA synthetase Binds to uncharged tRNA and activated itself to phosphorylate eIF2- Phosphorylation of eIF2 inhibits eIF2B GEF- With a phosphate, eIF2B binds eIF2 alpha with higher affinity and does not release ito Free eIF2 B decreases, translation initiation is inhibitedTernary complex on site recognition- YEAST:- If there’s a decrease in ternary complex levels, ribosome can begin scanning, but with noTC it won’t recognize the start site- TC may add on after scanning began and begin translating at a later start site- Gcn4 mRNA has unusually long 5’ UTR- 4 short upstream orfs before Gcn4 start site= uorfo Uorf= start codon, one codon, stop codon (3 codons total)o Uorfs 1 and 4 are needed, most importanto lacZ reporter fusion repressed and derepressed (starvation) WT= 100 to 600 Uorf-= 3000 to 5000 Uorfs regulate gcn4—repress translation- Gcn2 is required for Gcn4 expressiono Gcn2—shows that gcn2 is needed for derepression but not for general translationo Uorf1 unloads ribosome from TC because eIF2-GTP releases and ribosome scans without TCo If TC is abundant, TC reloads before uorf 4 and uorf 4 is translated No time to reload again before Gcn4 no Gcn4 translationo If low TC, ribosome bypasses uorf4 and if loaded before Gcn4, will translate Gcn4 Gcn4 translation decrease in global translation- High stress no translation AND no Gcn4- MAMMALS:- Mammals have 4 kinases that phosphorylate eIF2 alpha in response to stresseso HRI low heme, oxidative stresso PKR viral infection, dsRNA dsRNA dignals presence of viral infection and then cell apoptosiso PERK ER stress, unfolded proteinso Gcn2 low amino acids, uncharged tRNA, downregulation of translation- How do mice select what they eat in these conditions?o When given food with amino acids and without, mouse starving for amino acids knows to eat the food with amino acidso When Gcn2 mutant mouse, it does not know which food to eat- Alcohol derivatives of essential amino acids are competitive inhibitors of amino acid tRNA synthetaseo Binds active site and prevents tRNA synthetase from charging tRNA- Mouse on minimal (complete but not rich) threonin dieto The more time spent in from of the food (20 min to 40 min), the more food it ateo Inject control and inject threoninol to decrease tRNA charged in the brain After 40 minutes, the mouse does not eat much more than it did at 20 minuteso With D amino acid, doesn’t fit into the active site, so it won’t serve as an inhibitor no impacto At a high threonine diet, not much difference in feeding response- Is this behavior modulated by Gcn2 phosphorylation of eIF2?o Gcn2 +/+ knows difference in food/dieto GCn-/- does not know difference and even eats more of the improper diet- ATF4 in mammals instead of
View Full Document
Unlocking...