Shirley's Notes Molecular Biochem II (Ben)

Question 1

How are proteins made more fit for transport into the ER after translation?

Answer 1

HSP60

• temporarily folds them using ATP to keep their hydrophobic domains from interfering with membrane transport

Question 2

Describe the process of cotranslational translocation of proteins to the rER.

Answer 2

• Signal Recognition Particle (SRP), a protein-RNA complex of 6 PP chains binds ER signal sequences and the ribosomal A site
• Translation pauses + ribosome goes to rER to bind to SRP Receptor
• Protein translocator channel opens, translation restarts and growing PP chain enters rER lumen

Question 3

Where do chaperone proteins send misfolded proteins?

Answer 3

to cytosolic proteosomes for degradation

Question 4

What process of functional group addition helps proteins to fold properly?

(2 types of the process + where they happen)

Answer 4

Glycosylation - addition of sugar moieties

• N-glycosylation - addition to Asn amino group in the ER
• O-glycosylation - addition to Ser/Thr -OH grp in Golgi

Question 5

What chaperone protein helps assure proper glycosylation?

Answer 5

Calnexin is an integral ER membrane protein which retains unfolded N-glycoproteins in the ER.

It binds glycoproteins which have only 1 glucose in their oligosaccharide chain instead of 3.

If they fold correctly, they can leave. If not they will get more glucose added to them.

Question 6

What is retrograde translocation in reference to protein transport?

Answer 6

ER resident proteins have a “KDEL” retention sequence of 4 AAs at their C terminal.

Both ER and Golgi have luminal membrane receptors for this:

• ER receptor retains proteins w/ KDEL
• Golgi translocates them back to the ER where they belong.

Question 7

Which proteins mediate vesicle –> membrane fusion and how?

Answer 7

SNARE proteins

• vesicular v-SNARE binds target membrane t-SNARE with the help of G protein Rab’s GTP hydrolysis

Question 8

Which proteins direct vesicles to their desired targets?

With the help of what?

Answer 8

Coat proteins…

• COP-I - directs Golgi -> ER transport
• Clathrin - directs Golgi -> lysosome or R-mediated endocytosis
  
  • ARF hydrolyzes GTP to help budding of vesicles off of Golgi
• COP-II - directs ER -> Golgi transport
  
  • Sar-I hydrolyzes GTP to help budding off of ER

Question 9

Briefly describe structure and function of proteasomes.

Answer 9

• 28 unit

Question 10

Describe the process of ubiquitination.

Answer 10

1. E1 ubiquitin-activating enzyme - receives and activates ubiquitin using ATP (releasing AMP)
2. E2 ubiquitin-conjugating enzyme - gets ubiq. from E1
3. E3 ubiquitin ligase - recognizes ubiq. substrates via AA sequences + helps E2 transfer ubiq. to a Lysine residue on them

Question 11

How are proteins sent to lysosomes from the Golgi?

Answer 11

Mannose-6-phosphate is added to them.

• M-6-P receptors on Gogli membrane bind the protein and then send it in a vesicle to lysosome (retaining the M6P)
• ( P-mannosylation done via N-acetyl-glucosamine-P attachment to mannosylated protein from ER + subsequent removal of all but the mannose-P part )

Question 12

Describe nuclear import/export.

Answer 12

Import:

• Importins** (α protein-binding + β nuclear pore-binding) recognize **nuclear localization signals (NLS) (basic AAs)
• Ran-GDP incr. importin-NLS affinity in cytosol and GEF** exchanges for **Ran-GTP in nucleus which decr. affin. and drops off protein

Export:

• Opposite… exportins**… **nuclear export signal (NES)**… **GAP exchanges GTP->GDP

Question 13

Describe mitochondrial matrix targeting of proteins.

Answer 13

• N-terminal 20-50 basic AA sequence targets matrix
• HSP70 keeps protein unfolded + sequence exposed (using ATP)
• HSP70 takes protein to TOM translocase on outer membrane which binds signal sequence and sends protein thru another TOM pore
• TIM on inner membrane allows (+) charged sequence thru to matrix (via H+ gradient)

Question 14

Which bases are methylated on DNA and what is the result?

Answer 14

Methylation of cytosine results in blocking of transcription

(used as regulation mechanism)

Question 15

How can initiation factors be implicated in translation regulation?

Answer 15

Phosphorylation of eIF-2 reduces overall translation

• this allows cells to conserve resources + sometimes induce apoptosis

Question 16

What are 4 mechanisms of mRNA degradation control?

Answer 16

1. Degradation Sequences - usually near 3’ end, code for earlier degradation
2. Untranslated Regions - can create 3’ end fold instead of polyA tail + increase mRNA lifetime
3. Autoregulation - as in tubulin, when many tubulins molecules are present they activate a nuclease which binds the tubulin mRNA and degrades it
4. Nutrient Availability - when iron levels are high transferrin receptor mRNA degrades faster, when iron is low, opposite

Question 17

What important protein binds the TATA box region of the promoter?

What is its effect?

Answer 17

TF II-D

(transcription factor II D)

• coordinates the start of transcription

Question 18

What are 4 different DNA binding motifs on transcription factors?

Answer 18

1. Helix-turn-Helix - dimer, 3 helical regions separated by short turns
2. Zinc Finger - Cys/His residues bind zinc ion making finger-like chain which rests in DNA groove
3. Leucine Zipper - Leu residues at every other turn of alpha-helix forming “zipper” with Y-shaped region grabbing DNA
4. Basic Helix-loop-helix - two α helices connected by loop

Question 19

What are the two “types” of chromatin in relation to chromatin condensation?

Which is transcriptionally active?

Answer 19

Euchromatin - less condensed + transcriptionally active

Heterochromatin - condensed / inactive

Question 20

How do translational repressor proteins work?

Give an example.

Answer 20

By binding mRNA and blocking its attachment to the 40s ribosomal unit.

• ex: aconitase** binds **ferritin mRNA when iron not present
  
  • ferritin mRNA has “iron responsive element” and aconitase is “iron responsive element-binding protein”