Biochemistry - Final Exam

How does chloramphenicol work ? What does it target ?

This blocks the peptidyl transferase reaction on ribosomes. It interfers with aminoacyl-tRNA binding to the A site of the sibosome.

How does erthromycin work ? What does it target ?

This binds in the exit channel of the ribosome and thereby inhibits elongation of the chain

How does tetracyclines work ? What does it target ?

This blocks binding of aminoacyl-tRNA to A-site

How does strepromycin work ? What does it target ?

This prevents the transition from translation initiation to chain elongation and also can cause miscoding

How does cycloheximide work ? What does it target ?

This blocks the translocation reaction on ribosomes in eukaryotes

How does Ricin work ? What does it target ?

This causes a disulfide reduction, releasing the A subunit, which inactivates the large subunit of ribosomes. One ricin molecule can kill a cell.

How does Puromycin work ? What does it target ?

This inhibits the A site of ribosomes, reacting with the peptidyl-tRNA in the P site. This causes premature termination in both bacteria and eukaryotes

What are the major types of posttranslational modifications ?

Phosphorylation, Methylation, Acetylation, Ubiquitination, Glycosylation, Lipid modification

What ribosomomal subunits do Prokaryotes have ?

50S and 30S

What ribosomomal subunits do Eukaryotes have ?

60S and 40S

What is a Shine-Dalgarno sequence ?

This lines up mRNA so that tRNA can initiate

What site in Eukaryotes recognizes ALL three stop codons ?

eRF1

What does phosphorylation of a protein do ?

What does methylation of a protein do ?

What does acetylation of a protein do ?

What does ubiquitination of a protein do ?

What does glycosylation of a protein do ?

This increases solubility and protects secreted proteins from degradation and non-specific interactions.

What does lipid modification of a protein do ?

What site on the 30S subunit interacts with mRNA ?

IF-1 and IF-3

What ribosomal site interacts with fMET-tRNA ?

IF-2

What loss leads to the binding of the 50S subunit ?

Loss of IF-3

What does GTP hydrolysis of the 30S Subunit do ?

Releases IF1 and IF2 factors, results in the initiation.

What factors make up the Eukaryotic pre-initiation complex ?

eIF3, eIF1A, eIF5, eIF2-GTP-Met-tRNA

What factors make up the Eukaryotic cap binding complex ?

PABP, eIF4G, eIF4E, eIF4A, mRNA

Which factor binds the tail in Eukaryotes ?

PABP

What factor facilitates the release of the others and the addition of the 60S subunit via GTP hydrolysis ?

eIF5B

Which factor binds the cap in Eukaryotes ?

eIF4E

What are the basic steps for initiation in Eukaryotes ?

mRNA is bound to, Scanning for start codon (powered by ATP), GTP hydrolysis, 5B-GTP binds and recruits 60S, GTP-5B release, 1A release

What factor binds aminoayl-tRNA and GTP ?

EF-Tu

What are the termination / release factors in prokaryotes ?

RF-1, RF-2, RF-3

What codons does RF-1 recognize ?

UAG and UAA

What codons does RF-2 recognize ?

UGA and UAA

What codons does RF-3 recognize ?

No codons, only release factors

What is the termination factor in Eukaryotes ?

eRF

What codons does eRF recognize ?

All

What is ERAD ?

When a protein is misfolded, it will undergo a process known as ERAD (ER-Associated Degradation)

What does Hrd1 do in ERAD ? What is the mammalian homolog ?

hHrd1 is the homolog. This protein is an ERAD-M and-L E3 Ligase.

What does Doa10 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog Teb4. This protein is an ERAD-C E3 Ligase.

What does Ubc6 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog UBC6. This protein is an E2 Ubiquitin conjugating Enzyme.

What does Ubc7 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog UBC7. This protein is an E2 Ubiquitin conjugating Enzyme.

What does Cue1 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog does not exist, E3 Gp78 is similar but not exact. This protein is an anchor for Ubc7.

What does Ufd2 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog UBE4A and UBE4B. This protein is an Ubiquitin Extension Enzyme.

What does Cdc48 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog p97. AAA ATPase involved in retrotranslocation.

What does Der1 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog Derlin1. This protein is involved with retrotranslocation of ERAD-L substrates and anchors and recruits ERAD-M substrates to Hrd1.

What does PDI do in ERAD ? What is the mammalian homolog ?

Mammalian homolog PDI. It is a protein disulfide isomerase.

What does Kar2 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog Bip. It is a Luminal Hsp70, helps in recognizing misfolding.

What does Yos9 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog OS9. It is involved in the recognition and direction of ERAD-L substrates usually through glycosylation events.

What does Ydj1 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog Hsp40. This is a co-chaperone for Hsp70/Ssa1.

What does Ssa1 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog Hsp70. This is involved in the recognition of misfolded proteins.

What does Ufd1/NpL4 do in ERAD ? What is the mammalian homolog ?

Mammalian homolog is unknown. This is a cdc48 resident that recognizes Ubiquitinated substrates and helps direct substrates into Cdc48.

What does the Proteasome do in ERAD ? What is the mammalian homolog ?

Same as mammalian cells. It breaks down the misfolded protein.

What does PNG1 do in ERAD ? What is the mammalian homolog ?

Unknown homolog. It is a deglycosylase.

What does E1 protein do ?

It is the Ubiquitin-activating enzyme. It forms a thioester bond with the C-terminal Gly of the Ubiquitin.

What does E2 protein do ?

Ubitiquin is transferred to this protein on the Cys-thiol of E2. It then transfers the ubitiquin to the target protein with E3’s help.

What does E3 protein do ?

This selects protein for degradation by helping the transfer of ubitiquin to the Lys of the protein.

What proteins are involved in ERAD-L ?

PDI, Ufd1, Cdc48, Npl4, Ubx2, Usa1, Der1, Htm1/Mnl1, Yos9, Kar2, Jem1, Scj1, Hrd1, Hrd3, Hrd7, Cue1

What proteins are involved in ERAD-C ?

Ssa1, Ydj1, Hlj1 before moving to the ERAD-L complex

What proteins are involved in ERAD-M ?

ERAD-M are recognized by Der1, moving to the ERAD-L pathway

What motif signals going to the Nucleus ?

Positively charged

What motif signals going to the ER ?

Positive, hydrophobic, and polar

What motif signals going to the mitochondria ?

Positively charged amphiphillic alpha-helixes

What motif signals Nuclear Export ?

Leucine-rich

What protein is used in Prokaryotes to export other proteins ?

SecB and SecA with ATP

How are proteins secreted to the ER ?

SRP recognizes the motif and binds to it along with 2 GTP, stopping elongation, before binding to the translocon. After the hydrolysis of the GTP, the translocon opens at the signal motif is cleaved. This allows for elongation to to occur and the dissociation of 2GDP and SRP.

How is a protein glycosylated ?

Long carbohydrate chains (glycans) are synthesized on the membrane on a Dolichol-PP-Sugar by Glycosyltranferases. This is then transferred unto Asn/Ser/Thr residue via Oligosaccharide transferase.

How are proteins imported into the Nucleus ?

The protein is bound to by Importin on the NLS, transported through the NPC, and released. Ran-GDP is activated by GEF and helps release the Cargo and take the Importin back into the cytosol to be recycled. Ran is then activated by GAP.

What Amino Acids are O-linked for glycosylation ?

Ser and Thr

What Amino Acids are N-linked for glycosylation ?

Asn

How are proteins exported from the nucleus ?

The protein is bound to by Exportin on the NES, transported through the NPC, and released. Ran-GDP is activated by GEF and helps release the Cargo and take the Exportin-Target protein complex into the cytosol to be recycled. Ran is then activated by GAP.

How does Ran regulate transport of proteins ?

Ran regulates export by helping in moving the Exportin complex and Importin out into the Cytosol. It only goes back into the Nucleus once it is activated by GAP.

How does the decrease in sterol content in the ER result in the stimulation of Lipid synthesis ?

SCAP/SREBP is escorted to the Golgi following the targeting of Insig. There, two proteases release the regulatory domain of SREBP which is then transported to the nucleus.

What is metalation ?

The addition of metal ions.

What occurs if MTF-1 is highly saturated ?

This results in the expression of metalothionein (MT), which acts to shut down the Zinc transport channel as heavy and toxic metal ions are able to get through. This results in a general decrease in Zinc.

What family are typical Chaperones ?

Hsp70 Family

In Prokaryotes, what drives protein folding ?

GroEL is the two channel protein in which the protein is folded, and GroES act as a cap with 2ATP to prevent the protein from leaving until it is fully folded.

In Eukaryotes, how are proteins folded ?

Hsp40 binds to the polypeptide and brings to the ATP-Hsp70 complex. Following dissociation of Hsp40 and the creation of ADP, the complex is closed. This helps to

How does Hsp70 recognize proteins in need of folding ?

It binds to exposed hydrophobic segments

What happens when the protein is continunally misfolded ?

The chaperones will give up and degrade the protein.

How is Hsp70 recycled ?

NEF binds to the ATPase site and reactivates the ATP. This releases the protein and opens the complex.

What occurs when proteins are misfolded ?

Re-folding or degradation

What are SNARE proteins ?

These proteins mediate intracellular membrane fusion by pulling vesicles and target membranes together.

What is COP I ?

Inter-cisternal transport, helps in retrograde transport from CGN constitutive secretion

What is COP II ?

Anterograde transport from TER

What is Clathrin ?

Transport to lysosome/endosomes and secretory vesicles

How do mammalian cells cope with a build-up in misfolded proteins ?

Refolding, degradation, cell death

What protein stimulates Mitochondrial import ?

MSF

What protein is used in transporting proteins into the Mitochondria ?

TOM with ATP

What patch on a protein drives Ubiquitin ?

Hydrophobic patches

What is the difference between direct ubiquitination by E3 and mediated ubiquitination by E3 ?

Direct requires E3 ligase to act as a scaffold where E2 directly transfers the ubiquin to the protein. Whereas, mediated involved a two-step process where E3 forms a covalent intermediate.

What is the UPR response in the ER ?

When the ER is stressed, there is an accumulation of misfolded / unfolded proteins. This activates UPR and attempts to restore homeostasis. If this does not work, the cell dies.

What does the IRE1 pathway result in ?

ER chaperones, ERAD, Lipid synthesis
or
Apoptosis, Survival, Autophagy

What does the ATF6 pathway result in ?

Chaperones (BiP), ER quality control, Lipid Synthesis, XBP1

What does the PERK pathway result in ?

Apoptosis, Redox, Amino acid transport, Autophagy

What proteins are involved with IRE1 ?

Bip (Kar2), XBP1, RIDD, JNK, IRE1

What is the mechanism for the IRE1 pathway ?

Bip recognizes misfolded protein and is pulled away from IRE1. IRE1 then dimerizes and activates via phosphorylation on its RNAse domain. It then cleaves XBP1 mRNA, which re-ligates and is translated.

How does IRE1 recognize misfolding?

Direct interaction

What organisms have IRE1 ?

Yeast only

What is the mechanism for ATF6 ?

Under unfolded protein build-up, it is moved to the Golgi Body to be cleaved by S1P and S2P. Then, it is able to mobilize to activate UPR genes.

What is the mechanism for PERK ?

Upon stress, it dimerizes and phosphorylates itself and eIF2alpha. After, the eIF2-alpha is unable to function as a translation factor.

What protein encodes PP1C-De-phosphorylate ?

GADD34