Exam #1 Processes ONLY

Question 1

How does RNAi work?

Answer 1

1, double stranded RNA triggers RNAi

2. Dicer cleaves double stranded RNA—> siRNA
3. siRNA bound by Argonaute (RISC)—> one RNA strand cleaved & discarded
4. The single stranded siRNA bound to RISC directs RISC to the RNA produced by virus.
5. Exact match causes RISC to cleave target RNA—> degradation.

Question 2

How does Translation Initiation work?

Answer 2

1. mRNA binds to separate group of eIFs
2. 43S scans for AUG start codon on mRNA
3. 60S subunit joins the complex
4. Hydrolysis of two GTP forms complete 80S
5. Initiator tRNA binds to AUG at P site.

Question 3

How does Translation Elongation work?

Answer 3

1. amino acyl-tRNA brought to A site by Elongation Factors (EF)
2. GTP hydrolysis—> ribosome conformational change
3. Peptide bond formation between amino acids.
4. Hydrolysis of GTP—> Movement of the ribosome one codon
5. tRNA in P exits from E—> new amino acyl-tRNA comes in A.

Question 4

How does Translation Termination work?

Answer 4

1. Ribosome reaches a stop codon—> signals elongation to stop.
2. eRF1 (release factor) enters at A site and recognizes the stop codon.
3. eRF3 —> GTP hydrolysis—> cleaves the polypeptide chain from the tRNA at the P site.
4. mRNA dissocaties from ribosome and ribosome disassembles.

Question 5

What is miRNA mediated gene silencing and how does it affect gene expression at the translational level?

Answer 5

1. miRNA pairs with 3’UTR sequence: perfect match —> degradation, sort of match—> inhibition of translation
2. suppresses gene expression:
   - promotes mRNA deadenylation & degradation
   - inhibits initiation and elongation (translation)
   - degradation of nascent polypeptide

Question 6

What is protein ubiquitylation? How does it work?

Answer 6

It’s a mechanism for tagging proteins for proteosomal degradation:

1. Ubiquitin monomers are attached to lysine residues of proteins.
2. Cap proteins on proteosome recognize polyUbq-protein, removes Ubq and unfolds target protein.
3. Protesome digests the protein into small peptides
4. Peptides released to cytosol and degraded to amino acids.

Question 7

What are the steps of transport through the NPC?

Answer 7

1. Cargo protein with NLS binds to importin α/β in cytoplasm—> receptor-cargo complex.
2. receptor-cargo complex docks with the cytoplasmic filaments that extend from the NPC cytoplasm ring
3. receptor-cargo complex moves throug the nuclear pore by ingaging with FG domains of the FG-containing nucleoporins.
4. In the nuclear compartment, complex interacts with Ran-GTP—> disassembly and release of cargo.
5. Importin β is bound by Ran-GTP and shuttled back to the cytoplasm. Ran-GTP is hydrolyzed into GDP and transported back to the nucelus to be made into Ran-GTP again.
6. Importin α is bound to exportin and sent back to the cytoplasm.

Question 8

How does the K+ ion channel operate?

Answer 8

• K+ gate responds to LOW pH
• gating accomplished by conformational changes
• selects K+ over Na+, oxygen atoms of the carbonyl groups in the channel interact with K+ only.
• 2 carbonyl occupied at a time, no energy input needed

Question 9

What are steps the Na+/K+ pump undergoes?

Answer 9

** Each ATP hydrolyzed: 3Na+ out, 2K+ in

1. Pump in E1 (binding sites open) on cytoplasmic side. 3NA+ and ATP bound
2. occluded E1 (Na+ cannot flow back into cytosol)
3. Hydrolysis of ATP. E1—> E2
4. Binding sites open to extracellular, E2 releases 3Na+
5. Protein binds 2K+
6. Becomes occluded E2
7. Dephosporylation
8. ATP binds–> E1, 2K+ released
   * ** Note, ATP is not used to release K+. ATP just causes the conformational change. It is only used in releases Na+

Question 10

How are proteins synthesized in the RER?

Answer 10

1) Synthesis begins on free ribosomes—> produces nascent polypeptide–> signal sequence emerges—> SRP binds–> translation arrested.
2) SRP-ribosome binds to an SRP receptor on ER membrane.
3) Ribosome binds to translocon on ER—> SRP released—> signal protein binds to interior of translocon
4) contact between nascent polypeptide inside translocon–> plug displaced—> channel open –> Polypeptide enters ER lumen —> signal peptide is cleaved —> protein is folded

Question 11

How are the new proteins modified (processed) in the ER (as soon as it enters the RER cisterna)?

Answer 11

1. signal peptide is removed by signal peptidase.
2. Addition of carbohydrate (important to protein function and aids in proper folding)
3. Molecular chapertones in ER Lumen assist in proper folding
4. Protein processing enzymes in ER reduces cysteine residues on proteins entering lumen and join residues into disulfide bonds of proteins leaving ER.

Question 12

How are glycoproteins synthesized? (N-linked Glycosylation in the ER)

Answer 12

1) Lipid carriers (dolichol phosphate, DP) accept sugar molecules by glycosyltransferases
2) pre-assembled block of sugars is transferred from DP to certain asparagine residues on the nascent polypeptide. —> modifications—> glycoprotein

Question 13

What are the steps in Quality Control in the ER?

Answer 13

1) 2 of 3 glucose residues removed from protein
2) Bound by chaperone calnexin or calreticulin
3) Glucosidase removes final glucose to release from chaperone
4) motitoring enzyme UGGT recognizes misfolded proteins, adds back a glucose
5) protein re-enters cycle
6) If correct, protein exits cycle
7) If after many tries it is still not, it is destroyed.

Question 14

How does the UPR work?

Answer 14

1) ER has sensor proteins kept inactive by BiP
2) High levels of misfolded proteins—> pulls BiP from sensors—> activation of UPR
3) dimerization of sensor (PERK) –> becomes acivated protein kinase —> phosphorylates eIF2
4) phosphorylated EIF2 is inactive —> inhibition of protein synthesis.

Question 15

What is the alternate mechanism of UPR?

Answer 15

1) ER has sensor proteins kept inactive by BiP

2)High levels of misfolded proteins—> pulls BiP from
sensors—> activation of UPR

3) Release of BiP allows sensor (ATF6) to go to Golgi complex —> cytosolic domain of protein cleaved away
4) Cytosolic portion of sensor diffuses through cytosol—> nucleus
5) Stimulates the expression of genes to alleviate ER stress

Question 16

What is the vesicular transport model through the Golgi?

Answer 16

• Cargo move cis to trans in vesicles that bud from one membrane—> fuse with a neighbouring compartment farther along the stack.
• Anterograde (forward) movement

Question 17

What is the cisternal maturation model of the Golgi?

Answer 17

• Cisternae move cis to trans—> disperse at the TGN.
• Vesicles move cargo proteins in a retrograde (backgwards) direction.
• Enzymes that characterize each cisternae are sent back to lower cisternae —> large cargo carried along with moving cisternae up.

Question 18

How is the COPII coat assembled?

Answer 18

1) Sar 1 recruited to ER membrane by GEF (catalyzes GDP—> GTP)
2) Conformational change in Sar 1-GTP causes it to insert into cytosolic leaflet —> induces curvature in membrane
3) Dimer of 2 COPII proteins (Sec 23 & Sec 24) are recruited by Sar 1-GTP —> induces more curving.
4) Sec 24 interacts with ER export signals in cytosolic tails of cargo receptor proteins —> bind cargo on luminal side
5) Sec 13 and Sec 31 bind to form the outer cage of the protein coat.

Question 19

How is the COPII coat disassembled?

Answer 19

1) Hydrolysis of bound GTP —> Sar 1-GDP —> decreased affinity for the vesicle membrane
2) Dissociation of Sar 1-GDP is followed by the release of COPII proteins.

Question 20

How is the clathrin-coated vesicle assembled?

Answer 20

1) Like COPI, Arf 1 is involved in coat assembly

2) Coats are shed after budding and uncoated protein proceeds to destination

Question 21

How is the COPI coat assembled?

Answer 21

Arf 1 plays similar role as Sar 1 in COPII vesicles

Arf 1-GTP also needs to be hydrolyzed to GDP for disassembly of COPI coat.

Question 22

How do lysosomal proteins leave the Golgi complex?

Answer 22

Lysosomal enzymes are made in ER and carried to Golgi

1) In Golgi, mannose residues on lysosomal enzymes are phosphorylated
2) mannose 6-phosphate acts as a sorting signal recognized by MPRs on the TGN membrane which are then incorporated into clathrin vesicle.
3) mannose 6-phosphates interact with lysosomal enzyme on luminal side and the adaptors on the cytosolic side
4) Clathrin coat disassembles and the MPRs dissociate with their ligands
5) MPRs return to TGN
6) lysosomal proteins proceed to endosomes and then lysosomes
7) MPRs on PM capture lysosomal enzymes in the extracellular space and set them on the right path to the lysosome.

Question 23

How does membrane fusion work?

Answer 23

GTP-Rab proteins on vesicle and target membrane recruit tethering proteins

1) Vesicle docks via interactions between v-SNARE (vesicular) and t-SNARE (target)
2) possible transition state
3) Fusion complete, SNAREs reside on same membranes
4) Fusion pore opens allowing dischage of material

Question 24

How does autophagy work?

Answer 24

1) organelle is surrounded by double membrane (phagophore)
2) outer membrane of phagophore fuses with lysosome—> becomes autolysosomes—> leads to degradation
3) After digestion, becomes a residual body
4) residual body exocytosed or resides in cytoplasm as a pigment granule

Question 25

How does receptor-mediated endocytosis work?

Answer 25

1) Proteins that are to be imported into cell become concentrated on the extracelular surface of an indented region of PM, which forms the coated pit. 2) Coated pit sinks inward to create a coated bud. 3) Dynamine assembles into a helical collar around the neck of an invaginated coated pit 4) GTP hydrolysis causes twisting of the dynamin helix---> separation from the PM---> coated bud pinches off as a vesicle.

Question 26

How does the uptake of proteins into peroxisomes work?

Answer 26

- Peroxisomal proteins have a peroxisomal targeting signal: PTS for peroxisomal matrix protein or mPTS for peroxisomal membrane protein. - PTS receptors bind to peroxisome-destined proteins in the cytosol and shuttle them back to the surface of the organelle and pulls through through the membrane.

Question 27

What are the key reactions of glycolysis?

Answer 27

1. Gulocose (6-C) sugar is phosphorylated and rearranged to form 6-C biphosphate. 2. 6-C biphosphate is split into 2 3-C monophasphates 3. 3-C monophosphates are oxidized and the electrons lost reduce NAD+ to NADH (contains hiE electrons) 4. The phophate groups on carbon is transferred to ADP ---> ATP 5. Produces ATPs per gluocose oxidized and 2 pyruvate molecules.

Question 28

What happens to the pyruvates produced in glycolysis?

Answer 28

In the presence of O2: 1) moves to the mitochondrial matrix via membrane transporter 2) Reacts with Coenzyme A ---> acetyl CoA + CO2 + NADH through an irreversible exergonic, oxidative decarboxylation reaction. 3) Acetyl CoA enters TCA cycle.

Question 29

What are the key features of the TCA cycle?

Answer 29

1) 2-C acetyl group from acetyl CoA condenses with 4-C oxaloacetate ---> 6-C citrate 2) In subsequent reactions, citrate converted back into oxaloacetate, loses 2CO2 molecules in the process. 3) In subsequent reactions, succinyl CoA is produced 4) succinyl CoA is coupled to synthesis of one GTP by substrate level phosphorylation 5) In each turn of the cyle 4 pair os electrons are removed from C atomes ---> forms 3 NADH and 1 FADH2

Question 30

How is NADH and FADH2 converted to ATP in the ETC?

Answer 30

1) Electrons from NADH and FADH2 enter the ETC. 2) Electrons move to complexes---> releases energy--> translocates proteins to the intermembrane space 3) Proton gradient is established in the inner membrane 4) proton gradient drives the ATP synthase 5) ATP is formed

Question 31

How are integral proteins synthesized?

Answer 31

* SRP & SRP receptor interactions happen as before 1) Nascent polypeptide enters translocon ---> hydrophobic segment blocks translocation through 2) If charges align, Lateral gate opens---> transmembrane segment moves into the membrane 3) protein forms with N terminus in lumen of ER (positive end in cytosol) OR 2a) If charges don't align, translocon re-orients the transmembrane segment according to charges 3a) translocon opens ---> segments move into membrane 4a) protein with C-terminus in lumen of ER (positive end in cytosol)