Lecture 7 and 8

Question 1

Shine Dalgarno sequence

Answer 1

mRNAs have a 6-nucleotide Shine-Dalgarno sequence upstream of the AUG start codon.
it correctly positions AUG in the ribosomes and provides translational control mechanisms.

Question 2

Translational control mechanisms

Answer 2

1. A specific RNA-binding protein blocks access to the Shine Dalgarno sequence.
2. Temperature-regulated RNA structures - stem-loop of RNA structure blocks access to shine Dalgarno at lower temperatures.
3. Riboswitch (S-adenosyl methionine) - small molecules cause a conformational change to RNA blocking Shine Dalgarno sequence
4. Antisense RNA -it base pairs with mRNA and blocks SD

Question 3

Antisense RNA mechanism example

Answer 3

Iron Storage proteins

Question 4

Riboswitch mechanism example

Answer 4

S-adenosyl methionine

Question 5

Temperature regulation mechanism example

Answer 5

virulence genes of human pathogen Listeria monocytogenes

Question 6

eIF2

Answer 6

1. plays a crucial role in translation initiation
2. eIF2 forms a complex with GTP and recruits initiator tRNA (methionyl) to the small ribosomal subunit
3. The small ribosomal subunit binds the 5’ end of mRNA and scans for the first AUG
4. when AUG is recognized, eiF2, hydrolyzes GTP to GDP
5. GTP hydrolysis causes a conformational change in eiF2
6. eIF2 bound to GDP is released and inactive

Question 7

Reactivation of eIF2

Answer 7

it requires eIF2B which is a guanine nucleotide exchange factor (GEF) - it causes the exchange of GDP for GTP

Question 8

How is eIF2 reactivation regulated

Answer 8

phosphorylation
1. phosphorylated eIF2 sequesters eIF2B as an inactive complex
2. since there is more eIF2 than eIF2B in cells, all eIF2B is sequestered and translation is dramatically reduced.

Question 9

What happens when eIf2 is phosphorylated?

Answer 9

There is no exchange of GDP and GTP

Question 10

What steps do proteins undergo to become functional?

Answer 10

1. they must fold properly to adopt their 3D structure
2. they are covalently modified with chemical groups (eg. sugars, phosphate)
3. They interact with other proteins and small molecules (cofactors)

Question 11

Protein folding

Answer 11

Hydrophobic amino acids are buried in the interior core.

Question 12

When does the folding of proteins begin?

Answer 12

For some proteins, folding begins as they emerge from ribosomes, some are completely folded after synthesis

Question 12

hsp60 nd hsp70

Answer 12

1. they both interact with exposed hydrophobic residues of misfolded proteins.
2. they use energy from ATP hydrolysis to promote proper folding.
   hsp70 works before hsp60
   hsp70 binds to protein as it emerges from the ribosome - help fold it. there can be correctly and incorrectly folded proteins.
   hsp 60 - double barrel shape
   incorrectly folded protein will bind to the sites in the barrel. it will use ATP. Groes cap will come on, and it will give protein more time to fold.

Question 13

Chaperones for protein folding

Answer 13

many chaperones are called heat shock proteins since they are synthesized to high amounts by cells at elevated temperatures - as proteins begin to get denatured at higher temps

Question 14

Proteasome

Answer 14

Protein degrading apparatus

Question 15

What do exposed hydrophobic residues do?

Answer 15

they mark protein for degradation by the proteasome, competes with chaperones for misfolded proteins

Question 16

Where is the proteasome found?

Answer 16

cytosol and nucleus

Question 17

Describe the structure of the proteasome

Answer 17

It is a hollow cylinder with a cap at each end and an active site in the core, the cap protects cellular proteins from degradation

Question 18

What proteins does proteasome act on?

Answer 18

The proteasome acts on proteins that have been marked for destruction by the addition of a small protein tag named ubiquitin

Question 19

Ubiquitin-conjugating system

Answer 19

it is made up of three enzymes and it adds ubiquitin to proteins

Question 20

Three enzymes in the ubiquitin conjugating system

Answer 20

E1, E2 and E3

Question 21

E1

Answer 21

an ATP-dependent ubiquitin-activating enzyme creates an activated E1-bound ubiquitin

Question 22

E2 and E3

Answer 22

E2 is a ubiquitin-conjugating enzyme that accepts ubiquitin from E1 and exists as a complex with E3, a ubiquitin ligase that selects substrates

Question 23

process of addition of ubiquitin

Answer 23

1. ubiquitin gets added to E1, ATP gets converted to AMP
2. E2 binds to E1
3. E2 accepts ubiquitin from E1
4. E3 nd E2 will form a complex
5. E3 binds to specific degradation sequences in substrates
6. Ubiquitin is added to a lysin residue on the target protein
7. this process is repeated until a polyubiquitin chain is formed

Ubiquiting does NOT go on E3

Question 24

function of monoubiquitylation

Answer 24

histone regulation

Question 25

multiubiquitylation

Answer 25

endocytosis

Question 26

polyubiquitylation

Answer 26

proteasomal degradation + DNA repair

Question 27

How can E2 - E3 complex get activated

Answer 27

Phosphorylation by protein kinase, allosteric transition caused by ligand binding, allosteric transition caused by protein subunit addition

Question 28

how can a degradation signal get activated?

Answer 28

Phosphorylation by protein kinase, unmasking by protein dissociation, creation of destabilizing N terminus

Question 29

Protein Kinase A mediated gene exoression

Answer 29

Numerous extracellular stimuli result in increased levels of small molecule cyclic AMP (cAMP) - Activates protein kinase A (PKA)

Question 30

what does inactive PKA have?

Answer 30

2 regulatory subunits and 2 catalytic subunits

Question 31

What happens when cAMP binds to regulatory subunits?

Answer 31

Binding of cAMP to the regulatory subunits causes a conformational change and release of the active catalytic subunits

Question 32

What does PKA substrate include

Answer 32

PKA substrates include enzymes involved in glycogen metabolism in skeletal muscle and liver

Question 33

glycogen metabolism - pka

Answer 33

Ligand = adrenaline (epinephrine) * Response = to promote glucose release * Activated PKA has 2 effects: 1. promote breakdown of glycogen 2. inhibit glycogen synthesis * Glycogen is broken down into glucose-1- phosphate

Question 34

where is the inactivated PKA located?

Answer 34

cytosol

Question 35

what do PKA catalytic subunits phosphorylate

Answer 35

specific substrate proteins  activation of target genes with cAMP Responsive Elements (CRE)

Question 35

where does activated pka units translocate

Answer 35

nucleus

Question 36

how do protein interactions provide insight into function

Answer 36

guilt by association eg. DNA damage response network

Question 36

Activation of target genes with cAMP Responsive Elements (CRE):

Answer 36

- Activated PKA phosphorylates CREB (CRE Binding protein) * CREB recruits CBP coactivator (CREB Binding Protein) * Target genes are transcribed

Question 37

Ferritin RNA release mechanism when iron starvation

Answer 37

Cytosolic aconitase will bind to ferritin RNA near the start site and block translation

Question 38

Ferritin RNA release mechanism when excess RNA

Answer 38

Cytisikic aconitase will bind to iron, there will be a conformational change and the ferritin RNA is released.