Lecture 4.

Question 1

function of translational control mechanisms in eukaryotes and prokaryotes

Answer 1

both prokaryotes and eukaryotes use translational control mechanisms to regulate protein expression, often in response to stressful situations such as low nutrients, infection, or environmental stresses (eg temperature)

Question 2

describe prokaryotic translation

Answer 2

• mRNAs have a six nucleotide Shine-Dalgarno (SD) sequence in the 5’ UTR, upstream of the AUG start codon
• correctly positions AUG in the ribosome and provides translational control mechanisms

Question 3

4 mechanisms of transactional regulation in prokaryotes

Answer 3

1. a specific RNA binding protein blocks access to the SD sequence
2. temperature regulated RNA structures (eg virulence genes of human pathogen Listeria monocytogenes)
3. Riboswitch (eg S-adenosyl methionine)
4. antisense RNA (eg iron storage proteins)

Question 4

1. a specific RNA binding protein blocks access to the SD sequence

Answer 4

no translational repressor protein (RNA binding protein): gene is on > protein made

translational repressor protein (RNA binding protein): gene is off > no protein made

Question 5

2. temperature regulated RNA structures

Answer 5

low temperatures: RNA folds into a step-loop RNA structure that blocks access to SD > prevents ribosome binding > translation is inhibited

high temperatures: RNA structure melts/unfolds > SD sequence accessible to the ribosome > translation is initiated

Question 6

3. Riboswitch

Answer 6

regulatory segment within an mRNA (5′ UTR) that can bind a small molecule ligand and change its structure to control gene expression.

no small molecule: gene expression on

small molecule: causes structural rearrangement of RNA, blocking SD > gene expression off

Question 7

4. Antisense RNA

Answer 7

no antisense RNA: gene expression on

antisense RNA: produced elsewhere in the genome and base-pairs with mRNA, blocking SD > gene expression off

Question 8

do eukaryotes have shine-dalgarno sequences?

Answer 8

no, but there are similar mechanisms

Question 9

describe the translational regulation of iron

Answer 9

ferritin binds iron and releases it in a. controlled manner:
- not needed when iron is low: aconitase binds to the ferritin RNA near the start site and blocks translation
- translated when iron is in excess: aconitase binds iron, causing a conformational change, and leading to the release of ferritin RNA

Question 10

what does aconitase do in addition to controlling iron concentrations?

Answer 10

regulates transferrin receptor

Question 11

3 types of translational regulation in eukaryotes

Answer 11

1. repressor proteins can interfere with 5’ cap and 3’ poly-A tail interactions required for efficient translation
2. small RNA molecules can also regulate eukaryotic translation (miRNAs) via a different mechanisms than in prokaryotes
3. regulation of eukaryotic initiation factors (eIFs)

Question 12

eIF2

Answer 12

Plays a crucial role in translation initiation:

1. eIF2 forms a complex with GTP and recruits the initiator tRNA (methionyl) to the small ribosomal subunit
2. the small ribosomal subunit binds the 5’ end of mRNA and scans for the first AUG
3. when AUG is recognised, eIF2 hydrolyses GTP to GDP
4. GTP hydrolysis causes a conformational change in eIF2
5. eIF2 bound to GDP is released (inactive)
6. large ribosomal subunit comes in and translation occurs

Question 13

reactivation of eIF2 - normal conditions

Answer 13

requires eIF2B, which is a guanine nucleotide exchange factor (GEF), meaning it causes the exchange of GDP for GTP (NOT phosphorylation)

Question 14

reactivation of eIF2 - stress conditions (eg not enough nutrients)

Answer 14

1. specific protein kinases are activated and phosphorylate eIF2
2. since there is more eIF2 than eIF2B inc ells, phosphorylated eIF2 sequesters all eIF2B as an inactive complex
3. protein synthesis slows down dramatically

Question 15

how is translation resumed after sequestration of eIF2B by eIF2?

Answer 15

phosphates will desphosphorylate eIF2 and translation resumes

Question 16

are all mRNAs equally affected by eIF2?

Answer 16

no, not all mRNAs are equally affected

Question 17

describe the 3 steps required for proteins to become functional

Answer 17

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

Question 18

protein folding

Answer 18

• hydrophobic amino acids are buried in the interior core (not surface exposed)
• for some proteins, folding begins as they emerge from ribosomes; some are completely folded after synthesis

Question 19

why are chaperones important?

Answer 19

most proteins require chaperones for proper folding

Question 20

why are most chaperones called heat-shock proteins (Hsp)?

Answer 20

since they are synthesised to high amounts by cells at elevated temperatures

Question 21

describe two main types Hsp

Answer 21

Hsp70 and Hsp60 (chaperoning) assist protein folding:
- both interact with exposed hydrophobic residues of misfolded proteins
- both use energy from ATP hydrolysis to promote proper folding

Question 22

Hsp60

Answer 22

proteins that have been incorrectly folded enter the hsp60 complex (including a GroES cap) which acts as an isolation chamber to give it time to properly fold

Question 23

why is proper protein folding important?

Answer 23

• improperly folded proteins can aggregate and become toxic to cells
• misfolded proteins are the cause of many inherited human diseases

Question 24

how is improper protein folding prevented?

Answer 24

- the process is closely monitored by the proteasome (a protein degrading apparatus) - exposed hydrophobic residues mark protein for degradation by the proteasome, which competes with chaperones for misfolded proteins - the longer a protein takes to fold, the greater the chance of being degraded

Question 25

define the proteasome

Answer 25

an abundant protein complex found in the cytosol and nucleus (~1% of cellular protein)

Question 26

describe the structure of the proteasome

Answer 26

- a hollow cylinder with a cap at each end and an active site in the core - caps protect cellular proteins from degradation

Question 27

what does the proteasome act on?

Answer 27

proteins that have been marked for destruction by the addition of ubiquitin (a small protein tag)

Question 28

how is ubiquitin added to proteins?

Answer 28

by a ubiquitin-conjugating system made up of three enzymes

Question 29

three enzymes of ubiquitin conjugating system

Answer 29

E1: an ATP-dependent ubiquitin-activating enzyme. creates an activated E1-bound ubiquitin E2: ubiquitin-conjugating enzyme. accepts ubiquitin from E1 and exists as a complex with E3 E3: a ubiquitin ligase that selects substrates the E2-E3 complex works together

Question 30

how are ubiquitinated proteins ultimately destroyed?

Answer 30

1. E3 binds to specific degradation sequences in substrates 2. ubiquitin is added to a lysine residue on the target protein 3. process is repeated to form a polyubiquitin chain 4. polyubiquitin chain is recognised by specific receptor in the proteasome

Question 31

what functions - other than destruction - can ubiquitin modifications have?

Answer 31

monoubiquitylation > histone regulation multiubiquitylation > endocytosis polyubiquitylation (linkage through lys 48 of ubiquitin) > proteasomal degradation polyubiquitylation (linkage through lys 63 of ubiquitin) > DNA repair

Question 32

functions of ubiquitin modification depends on

Answer 32

number of ubiquitin molecules and type of linkage

Question 33

give two examples of how the destruction of a protein by the proteasome can be regulated

Answer 33

1. activation of a ubiquitin ligase can be caused by: - phosphorylation by protein kinase - allosteric transition caused by ligand - allosteric transition caused by protein subunit addition 2. activation of a degradation signal can be caused by: - phosphorylation by protein kinase - unmasking by protein dissociation - creation of destabilising N-terminus

Question 34

3 steps by which proteins become functional

Answer 34

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

Question 35

can only one modification occur on a protein?

Answer 35

no; multiple modifications can occur on the same protein

Question 36

how is PKA activated?

Answer 36

- numerous extracellular stimuli results in increased levels of cAMP, which activates PKA - binding of cAMP to the regulatory subunits of inactive PKA causes a conformational change and release of the active catalytic subunits

Question 37

subunits of inactive PKA

Answer 37

- two regulatory subunits - two catalytic subunits

Question 38

what is the function of PKA?

Answer 38

- PKA substrates include enzymes involved in glycogen metabolism in skeletal muscle and liver - ligand = adrenaline (epinephrine) - response = to promote glucose release

Question 39

draw a flow chart for the effect of epinephrine to the signal molecule

Question 40

activated PKA has two effects:

Answer 40

1. promotes breakdown of glycogen: glycogen is broken down into glucose-1-phosphate (which is converted to glucose-6-phosphate > glycolytic pathway) 2. inhibits glycogen synthesis

Question 41

where are inactive and activated PKA located?

Answer 41

- inactive PKA is located in the cytosol - activated PKA catalytic subunits can also translocate to the nucleus

Question 42

how can PKA affect gene transcription?

Answer 42

PKA catalytic subunits phosphorylate specific substrate proteins, leading to the activation of target genes with cAMP Responsive Elements (CRE): 1. activated PKA phosphorylates CREB (CRE Binding protein) 2. CREB recruits CBP coactivator (CREB Binding Protein) 3. when CREB binds to the CRE, target genes are transcribed (eg in the liver, transcription of the gene for glucose-6-phosphatase, which dephosphorylates glucose-6-P to glucose > released into the blood )

Question 43

proteins usually function in

Answer 43

large multi-protein complexes composed of static and transient interactions

Question 44

define the interactome map

Answer 44

the complete collection of protein-protein interactions of an organism

Question 45

describe the interactome map

Answer 45

- only 8000 proteins - each dot is a protein node - each line is an interaction edge

Question 46

slides 20-23