what are translational control mechanisms in prokaryotes and eukaryotes used for
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 (e.g., temperature)
Prokaryotes:
what does the Shine-Dalgarno (SD) sequence do in prokaryotes for translational regulation?
mRNAs have a six nucleotide Shine-Dalgarno (SD) sequence
upstream of the AUG start codon
➢ correctly positions AUG in the ribosome and provides
translational control mechanisms
Outline Mechanism 1 of translational regulation in prokaryotes – using translation repressor proteins and the SD sequence
change represses
Mechanism 1: A specific RNA binding repressor protein blocks access to the SD sequence
ON:
SD sequence is before the AUG start codon –> protein is made until the stop codon
OFF:
SD sequence is before the AUG start codon –> translation repressor protein binds to SD sequence –> protein is not made
Outline Mechanism 2 of translational regulation in prokaryotes – using temperature to make the SD sequence more accessible
change activates
Mechanism 2: Temperature regulated RNA structures
e.g., virulence genes of human pathogen Listeria monocytogenes
OFF:
the SD sequence is on the 5’UTR loop and thus cannot code for protein
ON:
however, increased temperature unravels the 5’UTR so the SD sequence is more accessible and thus codes for protein
Outline Mechanism 3 of translational regulation in prokaryotes – small molecules and RNA rearrangement
Mechanism 3: Riboswitch e.g., S-adenosyl methionine
ON:
SD sequence is accessible and thus translation occurs.
OFF:
if specific small molecules are present it causes structural rearrangement of RNA so that the SD sequence is blocked and thus no coding
Outline Mechanism 4 of translational regulation in prokaryotes – antisense RNA
Mechanism 4: Antisense RNA e.g., iron storage proteins
ON:
SD sequence is accessible and translation occurs
OFF:
Antisense RNA produced elsewhere in the genome spontaneously base-pairs with mRNA where the SD sequence is located and thus and blocks SD for coding
how is translational regulation differ in eukaryotes from prokaryotes
- No Shine-Dalgarno sequences, but there are similar mechanisms
➢ translational repressors can bind near initiator AUG and inhibit translation
(e.g., aconitase)
Eukaryotes:
how does ferritin mRNA and translational repressors regulate translation in eukaryotes
(recall aconitase also regulates transferrin receptor) COMPARE L6
Ferritin binds iron and releases it in a controlled
manner
without iron:
➢ aconitase binds to the ferritin RNA near the start site and blocks translation
with iron:
➢ aconitase binds iron
➢ conformational change so that the aconitase releases from the mRNA
➢ ferritin RNA released and Ferritin is made and translated
what are the two additional ways translation can be regulated in eukaryotes from last class (post-transcriptional technically)?
- additional to ferritin example
- what is a new way we can regulate translation
(4 total)
- Repressor proteins can also interfere with 5’ cap and 3’ poly-A tail interactions required for efficient translation
- Small RNA molecules can also regulate eukaryotic translation (miRNAs)
➢ different mechanism than in prokaryotes - There are also other eukaryotic specific
mechanisms (e.g., eIF2)…
Translational Regulation: Eukaryotic Translation Initiation
how is there regulation of eukaryotic initiation factors (eIFs) for translational initiation?
regulation of the ribosomal unit (this lesson) which moves along the regulated prepped mRNA loop (last lesson). both regulation processes use eIFs
Regulation of eukaryotic initiation factors (eIFs) to regulate translation:
- eIF2 plays a crucial role in translation initiation
- eIF2 forms a complex with GTP and recruits the 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 on the complex - eIF2 bound to GDP is released
- eIF2 bound to GDP is inactive…
how do we get another round of translation with eIFs
1/2 things that can happen after one round of translation
- reactivation of eIF2 requires eIF2B which is a guanine nucleotide exchange factor (GEF), meaning it causes the exchange of GDP for GTP
- we have an inactive eIF2- GDP complex as an end product from eukaryotic translation initiation (1st round)
- guanine nucleotide exchange factor (GEF), eIF2B, binds to the inactive eIF2 complex
- this releases the GDP from the eIF2
- GTP is added to the eIF2 and the GEF is released
- NOTE THIS IS NOT A PHOSPHORYLATION ITS AN EXCHANGE
BUT eIF2 reactivation is regulated by phosphorylation
how is eIF2 reactivation regulated by phosphorylation
ie. how to shut down translation
2/2 things that can happen after one round of translation
shut down the little eiF2 through phosphorylation so that more protein is not formed (stop translation)
- phosphorylated eIF2 sequesters GEF eIF2B as an inactive complex
- since there is more eIF2 than eIF2B in cells, all eIF2B is sequestered and
translation is dramatically reduced - not all mRNAs are equally affected by eIF2 phosphorylation
if translation is shut down through phosphorylation, how do we reactivate trasnlation if nutrients are found
simply remove the phosphate group and restart
Post-Translational Regulation: Proteins
what are the three typical ordered steps for proteins to become functional
- proteins must fold properly to adopt their 3D structure
- proteins are covalently modified with chemical groups (eg. sugars, phosphate)
- Proteins interact with other proteins and small molecules (cofactors)
Post-Translational Regulation: Proteins
STEP 1 - proteins must fold properly
how are hydrophobic amino acids folded to make the proteins?
when does folding occur for some proteins vs others?
- hydrophobic amino acids are buried in the interior core (ie. not surface exposed)
- For some proteins, folding begins as they emerge from ribosomes; some are completely folded after synthesis
Post-Translational Regulation: Proteins
STEP 1 - proteins must fold properly
what are chaperone proteins and what are they also called?
- most proteins require a special class of proteins called chaperones for proper folding
- many chaperones are called heat-shock proteins (Hsp) since they are synthesized to high amounts by cells at elevated temperatures
Post-Translational Regulation: Proteins
STEP 1 - proteins must fold properly
how does Hsp70 and Hsp60 (chaperonin) used to assist in protein folding
- Both interact with exposed
hydrophobic residues of misfolded proteins - Both use energy from ATP hydrolysis to promote proper folding
Hsp70:
- Hsp70 binds with protein as it emerges from the ribosome
- uses ATP to correctly fold protein
- typically results in correct folding of protein, but if not it goes ot Hsp60 to get correctly folded
Hsp60:
- incorrect protein enters the double barrel on one side. this is because the misfolded protein has hydrophobic groups on the outside and needs to be entrapped to avoid contact with water. the barrel on the inside has hydrophobic binding sites. (only one barrel can work at a time)
- a cap comes and closes the double barrel on the side the protein entered
- Hsp60 acts as an isolation chamber and essentially gives the protein more time to evolve into the correct folding
- then once correctly folded, uses ATP to release correctly fold protein and remove the cap.
how does the proteasome help with regulating toxic improperly folded proteins
Improperly folded proteins can aggregate and become toxic to cells
* misfolded proteins are the cause of many inherited human diseases
* the process is closely monitored by a protein degrading apparatus called
the proteasome
* exposed hydrophobic residues mark protein for degradation by the
proteasome; competes with chaperones for misfolded proteins
- longer time to fold, more chance of being degraded
