Lecture 7 and 8 Flashcards
Shine Dalgarno sequence
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.
Translational control mechanisms
- A specific RNA-binding protein blocks access to the Shine Dalgarno sequence.
- Temperature-regulated RNA structures - stem-loop of RNA structure blocks access to shine Dalgarno at lower temperatures.
- Riboswitch (S-adenosyl methionine) - small molecules cause a conformational change to RNA blocking Shine Dalgarno sequence
- Antisense RNA -it base pairs with mRNA and blocks SD
Antisense RNA mechanism example
Iron Storage proteins
Riboswitch mechanism example
S-adenosyl methionine
Temperature regulation mechanism example
virulence genes of human pathogen Listeria monocytogenes
eIF2
- plays a crucial role in translation initiation
- eIF2 forms a complex with GTP and recruits initiator tRNA (methionyl) to the small ribosomal subunit
- The small ribosomal subunit binds the 5’ end of mRNA and scans for the first AUG
- when AUG is recognized, eiF2, hydrolyzes GTP to GDP
- GTP hydrolysis causes a conformational change in eiF2
- eIF2 bound to GDP is released and inactive
Reactivation of eIF2
it requires eIF2B which is a guanine nucleotide exchange factor (GEF) - it causes the exchange of GDP for GTP
How is eIF2 reactivation regulated
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.
What happens when eIf2 is phosphorylated?
There is no exchange of GDP and GTP
What steps do proteins undergo to become functional?
- they must fold properly to adopt their 3D structure
- they are covalently modified with chemical groups (eg. sugars, phosphate)
- They interact with other proteins and small molecules (cofactors)
Protein folding
Hydrophobic amino acids are buried in the interior core.
When does the folding of proteins begin?
For some proteins, folding begins as they emerge from ribosomes, some are completely folded after synthesis
hsp60 nd hsp70
- they both interact with exposed hydrophobic residues of misfolded proteins.
- 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.
Chaperones for protein folding
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
Proteasome
Protein degrading apparatus
What do exposed hydrophobic residues do?
they mark protein for degradation by the proteasome, competes with chaperones for misfolded proteins
Where is the proteasome found?
cytosol and nucleus
Describe the structure of the proteasome
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
What proteins does proteasome act on?
The proteasome acts on proteins that have been marked for destruction by the addition of a small protein tag named ubiquitin
Ubiquitin-conjugating system
it is made up of three enzymes and it adds ubiquitin to proteins
Three enzymes in the ubiquitin conjugating system
E1, E2 and E3
E1
an ATP-dependent ubiquitin-activating enzyme creates an activated E1-bound ubiquitin
E2 and E3
E2 is a ubiquitin-conjugating enzyme that accepts ubiquitin from E1 and exists as a complex with E3, a ubiquitin ligase that selects substrates
process of addition of ubiquitin
- ubiquitin gets added to E1, ATP gets converted to AMP
- E2 binds to E1
- E2 accepts ubiquitin from E1
- E3 nd E2 will form a complex
- E3 binds to specific degradation sequences in substrates
- Ubiquitin is added to a lysin residue on the target protein
- this process is repeated until a polyubiquitin chain is formed
Ubiquiting does NOT go on E3
function of monoubiquitylation
histone regulation
multiubiquitylation
endocytosis
polyubiquitylation
proteasomal degradation + DNA repair
How can E2 - E3 complex get activated
Phosphorylation by protein kinase, allosteric transition caused by ligand binding, allosteric transition caused by protein subunit addition
how can a degradation signal get activated?
Phosphorylation by protein kinase, unmasking by protein dissociation, creation of destabilizing N terminus
Protein Kinase A mediated gene exoression
Numerous extracellular stimuli result in increased levels of small
molecule cyclic AMP (cAMP)
- Activates protein kinase A (PKA)
what does inactive PKA have?
2 regulatory subunits and 2 catalytic subunits
What happens when cAMP binds to regulatory subunits?
Binding of cAMP to the regulatory subunits causes a conformational
change and release of the active catalytic subunits
What does PKA substrate include
PKA substrates include enzymes involved in glycogen metabolism in skeletal muscle and liver
glycogen metabolism - pka
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
where is the inactivated PKA located?
cytosol
what do PKA catalytic subunits
phosphorylate
specific substrate
proteins
activation of target genes with
cAMP Responsive Elements
(CRE)
where does activated pka units translocate
nucleus
how do protein interactions provide insight into function
guilt by association
eg. DNA damage response network
Activation of target genes with cAMP
Responsive Elements (CRE):
- Activated PKA phosphorylates
CREB (CRE Binding protein) - CREB recruits CBP coactivator
(CREB Binding Protein) - Target genes are transcribed
Ferritin RNA release mechanism when iron starvation
Cytosolic aconitase will bind to ferritin RNA near the start site and block translation
Ferritin RNA release mechanism when excess RNA
Cytisikic aconitase will bind to iron, there will be a conformational change and the ferritin RNA is released.
