SM 12 Flashcards
(17 cards)
What are the steps of translation-initiation?
The initiator tRNA binds eIF2 (a GTP binding protein), which will bind to the 40S subunit at the P site. The 5’cap of the mRNA binds to CBP, which contains 20 subunits including EIF4E. The binding of CBP recruits other TF. The mRNA-CBP complex assoc with the Methionyl tRNA-40S ribosome complex. An RNA helicase in an EIF subunit unwinds the secondary structure (hairpins) at the 5’end of mRNA using ATP hydrolysis and the ribosome complex scans the mRNA 5’ to 3’ until it reaches the AUG codon. The GTP bound to EIF2 is hydrolyzed and the initiation factors are released. This loosens the affinity of EIF2 to the initiator tRNA. This allows large subunit to bind long with EIFB (another GTP-binding protein). The large subunit binds with the methionyltRA bound to the ribosome at the P site. This will release the EIF2-GDP and the other initiator factors. Once those are released the EIFB will hydrolyze its GTP and this will allow ribosome to proceed and start translating.
What is the difference between normal genes and genes that have IRES (internal ribosome entry sites)?
They translate cap-independtly. Mostly involved in stress. Typical of viruses.
What are the steps of translation-elongation?
- During intitiation, a methionyl-tRNAiMet is bound to the P site on the ribosome. An aminoacyl-tRNA specified by the codon in the A (aminoacyl) site of the ribosome binds.
- The aminoacyl-tRNA is bound to an elongation factor EF1α containing bound GTP (EF1α is a heterotrimeric G-protein). Upon binding to the A site, GTP is hydrolyzed to GDP and the EF1α is released.
- Elongation occurs when the amino acid on the tRNA in the A site forms a peptide bond with the initiator methionine or peptide bound to the tRNA in the P site. The peptidyltransferase that catalyzes the bond formation is the rRNA of the large ribosomal subunit (a ribozyme). The growing peptide chain is now attached to the tRNA in the A site and the tRNA in the P site is uncharged.
- Translocation of the ribosome to the next codon requires another G-protein, elongation factor EF2. This elongation factor binds to the ribosome inducing a conformational change that moves the mRNA and bound tRNAs with respect to the ribosome. The spent tRNA that was in the P site is released and the peptidyl-tRNA now occupies the P site and the A site is unoccupied. The process repeats.
Describe process of translation - termination.
Termination of translation occurs when a stop codon enters the A site. No tRNAs bind to stop codons, instead a protein called release factor binds to the ribosome and cause hydrolysis of the peptide chain from the final tRNA. The polypeptide chain is released from the ribosome and the ribosomal subunits dissociate, releasing the mRNA.
How does heme regulate translation in RBC?
In RBCs globin synthesis is regulated by heme because we need to balance amount of globin chains with the heme. eIF2alpha (involved in priming small subunit of ribosome) is normally active unless phosphorylated. Free heme binds to and inhibits the heme-regulated inhibitor kinase that phosphorylates eIF2α, rendering it inactive. When heme levels are high (we need more globin), the inhibitory kinase is inactive, eIF2α is not phosphorylated and is active.
How does iron starvation vs excess iron affect ferritin translation.
Ferritin is an intracellular iron storage protein. Transferrin receptor takes up iron into the cell
Under iron starvation we want to take up as much iron as possible so we up regulate amount of transferrin receptor. But we do not need ferritin so we downregulate its synthesis. The ferritin mRNA has hairpin loop in its 5’UTR. Transferrin has hairpin in 3’UTR. Aconitase bind iron as part of their mechanism. So when no iron, cytosolic aconite is bound to ferritin mRNA so it can’t translate. binding of aconitase to 3’UTR of transferrin, stabilizes it so that it can be translated.
With excess iron, you want to stop too much iron from coming in so get rid of receptor, iron binds to acontiase, it is released from hairpin loop which gets cut by ribonuclease and message is degraded so no more receptor. The aconite releases the ferritin hairpin and allows translation of ferritin to occur
Regulatory proteins have long half-life. T/F.
F. They have short half-life.
How do we target Rapamycin mTOR for cell growth/proliferation?
Major regulator of cell growth is Rapamycin (an immunosuppressant) MTOR which responds to growth signals. It can be activated by growth factor stimulation which activates signal traduction pathway that forms PIP2 (formed by membrane phospholipid ionsitorl phospholipid) that activates mTOR kinase activity. mTOR will stimulate other factors that stimulate ribsome synthesis. Regulates ribosome activity by phos a part of small subunit called S6 by activating another kinase called S6 Kinase. It also reg initiation by regulating eIF4E by inhibiting protein called 43-binding protein (4E-BP). All to stimulate protein syntheis and growth.
How are targets for degradation ubiquinated?
E1 activates ubiq using ATP hydrolysis. It transfers the activated ubiq to E2 which then interacts with E3 adaptor which binds to protein that will be degraded and allow transfer of Ubiq to lysine residues on substrate. cap on proteasome recog the polyubiq chain which it will break up for re-use. The protein itself is unfolded so cap has chaperon like activity that can unfold the protein which is fed through channel, chopped up into AA that is released through other end. Functions in cytoplasm and nucleus.
How does lysosome attract drugs with negative charge?
The internal env is acidic so it attracts the drugs with negative charge.
Described ERAD.
If proteins made in the ER are misfiled they are retrotranslocated to cytosol where they are degraded by proteosomes.
What happens if too many mis-folded proteins or when it takes too long to fold the proteins (as with CFTR in cystic fibrosis)?
Triggers UPR. The chaperones in ER trigger IRE1, and ETF6 in the ER membrane. IRE 1 reg splicing of mRNA of gene regulatory protein (which usually remains incompletely spliced in nucleus), this protein will be made and will activate genes that make the proteins involved in folding of proteins in ER. PERK will phosphorylate and inactivate translation initiator factor to reduce number of proteins entering the ER and it will selectively translate another gene regulatory protein. ATF6 will activate a protease in the cytoplasm, clips part of the protein off the membrane which will also increase transcription of genes involved with folding in the ER.
What are amyloids?
Form by the alternative folding of a small number of proteins. The alternatively folded proteins polymerize into insoluble fibrous structures called amyloid.
How do aggregates form aggresome?
by assoc with dynein.
How does autophagy occur?
The autophagic vacuole forms when flattened sheets of membranes derived from the smooth ER coalesce to form phagophore which associates with LC3 which becomes cov linked to phosphatidyl serine in the membrane. This triggers it to engulf stuff. Then it will seal and become double membrane stucture. Fusion of the autophagic vacuole with lysosomes forms an autolysosome where the engulfed contents are degraded as well as inner membrane with the LC2. Ultimately this will break up releasing LC3 for reuse.
How does chaperone-mediated autophagy (CMA) direct transfer of proteins to lysosomes.
direct transfer of proteins into lysosomes through channels. Protein in lysosome membrane called LAMP2A. Chaperon recog proteins to be degraded, interacted with LAMP2A which aggregate in membrane and form channel to allow protein to come in and be degraded. These proteins have KFERQ sequence.
What is signal assoc with selective microautophagy?
hsc70
