Molecular FINAL

Question 1

NLS Composition

Answer 1

• Usually rich in Lys and Arg plus one proline.
• Monopartite, some times bipartitie

Question 2

Two stages of Active Nuclear Transport

Answer 2

• Docking
• Translocation

Question 3

Active Nuclear Transport:

Docking

Answer 3

• Binding to the nuclear pore
• Protein requires NLS
• Carrier proteins assist in docking

Question 4

Active Nuclear Transport:

Translocation

Answer 4

• Movement through pore
• Requires ATP

Question 5

NLS Experiment

Answer 5

1. Cells were treated with digitonin
2. Resulted in the cell membrane being permeabilized, not the nuclear envelope though.
3. Allowed for the study of the importation of the protein of interest.
   
   • A protein was then added with NLS and the location was monitored.

Question 6

Where does CBC go? and what does it need?

Answer 6

• Nuclear Cap Binding Complex for mRNA export
• CBC
  
  • Required export signal: NES
  • Carrier: Exportin-1

Question 7

Where do Nuclear Proteins go? and what does it need?

Answer 7

• Required import signal: NLS
• Required Carrier: Importin a, B

Question 8

Where do hnRNP proteins go? and what does it need?

Answer 8

• Required import signal: Mg
• Required Carrier: Transportin

Question 9

Where do Ribosomal Proteins go? and what does it need?

Answer 9

• Required import signal: (Unknown)
• Required Carrier: Importin B3

Question 10

Experiment that determined how the Nuclear pore accommodated bigger than 10 nm.

Answer 10

1. Gold particles (20 nm) that were covered in nuclear protein were injected into the cytoplasm.
   
   • Gold was used since it is electron dense and electromagnetic specturm.
2. They were then observed to accumulate at the pore sites.
3. They then were observed to be accumulated inside the nucleus.
   
   • This showed that the nuclear pore was able to expand to accomodate larger molecules.
     
     • This indicated a dynamic structure, and that they likely have a “Gating mechanism” to allow macromolecule passage.

Question 11

KDEL Signal Sequence

Answer 11

Lys-Asp-Glu-Leu in C-terminus

Question 12

KDEL Role

Answer 12

• Retains protein in ER
• Retrieves proteins that have “escaped” to the Cis - golgi

Question 13

Emerin function?

Answer 13

• Nuclear protein important for anchorage to the cytoskeleton interteracting with Lamin A.
  
  • Mutations of this protein is involved in Dreifuss-Emery muscular dystrophy.

Question 14

Describe Lamin Proteins

Answer 14

• Four different lamins (A, B1, B2, C)
  
  • These are fibrous proteins, similar to intermediate filaments
    
    • Often modified by addition of lipids (These promote anchoring into the inner layer of the nuclear membrane).
  • Modified by prenylation to facilitate lipid insertion into the nuclear membrane.
  • If any of these are mutated then the structure that forms on the inside of the cell may not be able to converse with itself properly.

Question 15

Stages of Active nuclear Transport?

Answer 15

• Docking
  
  • Binding to the nuclear pore
  • Protein requires NLS
  • Carrier proteins assist in docking
• Translocation
  
  • Movement through pore
  • Requires ATP

Question 16

Chromatin

Eu- vs. Hetero-

Answer 16

• Euchromatin:
  
  • Decondensed
  • Distributed throughout the nuclus
  • Dispersed (loops)
• Heterochromatin
  
  • Mostly localizes the periphery of the nucleus
  • 2 types
    
    • Constitutive
      
      • Always condensed, very rarely transcribed
        
        • Satellite sequences.
    • Facultative
      
      • Varies with cell type, or during differentiation.

Question 17

Chromosome localization experiment

Answer 17

• Cells were labelled with bromodeoxyuridine
• Incorporated into the newly replicating DNA
• Stain cells with antibody BrdU, showed presence of multiple foci
• These replication foci were then detected by immunofluorescence.

Question 18

snoRNP Structure and role

Answer 18

• snoRNA + 8-10 proteins
• snoRNP associates with fibrillar proteins and modifies rRNA.

Question 19

Translocon structure

Answer 19

• SRP + SRP receptor + Sec61 + signal peptidase

Question 20

Process of Co-translational ER targeting

Answer 20

E-I-A-R-C-R

1. Elongation: Elongating chain, signal sequence emerges from the ribosome
2. Interception: SRP binds, stops translation/elongation. Will recognize the SRP receptor bound to the ER pore through interactions with the Sec61 protein.
3. Arrival: SRP released and the signal sequences enters the translocation complex.
4. Resumption: Translation resumes and the signal sequence can move into the translocation complex towards the signal peptidase.
5. Cleavage: The signal sequence will then be cleaved.
6. Release: The protein is then released into the lumen of the ER.

Question 21

Modified by N-Linked glycosylation in ER Steps:

Answer 21

,

1. Modification: Residue is modified (acceptor is an asparagine)
2. Addition: N-acetyl glucosamine is transfered from a dolichol-phosphate residue and attached to the Asn.
3. Sugar Addition: Next the addition of Mannose (branched structure) followed by 3 Glucose residues.
4. Removal: The 3 glucose are removed and then one to four mannose are removed
   
   1. ER Glucosidase 1 and 2 will remove the glucose
   2. ER mannosidase will remove one to four mannose residues.
5. Completetion: The N-linked glycoprotein is now ready to be transferred to the Golgi for eventual further modifications.

Question 22

Clathrin Structure

Answer 22

• Triskelion structure
  
  • 3 heavy chains (190 kD)
  • 3 Light chains (~30 kD)

Question 23

dynamin

Answer 23

• 900 aa
• polymerized around neck of forming vesicle
• GTP is hydrolyzed to GDP
  
  • Regulates the “Contractions” of dynamin

Question 24

ARF

Answer 24

• ARF
  
  • ADP-Ribosylation factor
  • Myristoylated
  • ARF and coatomer formation for COP-1 vesicles
    
    • Provides binding sites for coat proteins to assemble and bind adaptors
      *

Question 25

COP-2 Vesicle budding

Answer 25

• Similar to ARF but done by Sar1

Question 26

V-Snare

Answer 26

Protein Carried by vesicle

Question 27

t-Snare

Answer 27

Protein on Target membrane.

Question 28

SNARE Hypothesis:

Answer 28

• Also need Rab Proteins to facilitate “tethering formation = GTP-binding protein
• NSF protein = ATPase
• SNAP protein = Binds to membrane
  
  • SNAP: Soluble NSF-attachment protein.
• Binding Regulated by Rab-GTP –> Rab GDP transition

Question 29

What are the composition, and function of the Signal Recognition Particle?

Answer 29

• SRP is a ribonucleoprotein containing:
  
  • 300 nt srpRNA or 7S RNA (acting as a necessary scaffold) plus 6 proteins.
• Role:
  
  • binding to signal sequence of peptide or protein,
  • arrest translation
  • bind to receptor protein on the ER. Srp RNA interacts with ribosome.

Question 30

Aminoacyl tRNA synthetases are enzymes that

Answer 30

attach amino acids to specific transfer RNAs.

Question 31

NFkB and IFkB

Answer 31

.Cytosolic protein can mask the NLS (NFkB’s NLS masked by IFkB preventing binding of importin a)

Question 32

Two important gene mutations for Acute myeloid leukemia

Answer 32

• SRSF2
  
  • Serine and Arginine rich splicing factor 2
  • Majority of splicing changes with SRSF2 mutations involved alternative splicing
• IDH2
  
  • Regulates splicing.
• If both are mutated SRSF2 can splice out IDH2 removing regulation.

Question 33

ESH2

Answer 33

• Histone-3-Lys-36-Tri-methyl
  
  • Slows down polymerase so it is more accurate.

Question 34

Why so many Rab proteins?

Answer 34

• They appear to be involved in specific targeting to particular compartments of the cell.