Lecture #11 (Translation #2) Flashcards
Broad Classes of Regulation
- Global Regulation
- Gene Specific Regulation
What does a Cell do in crisis
If a cell is in crisis (no energy in cell or no proteins in ell) = cell makes ribosomes and translates thnigs
- Ribosomes are the major consumer of energy in the cell
- Bad solution = for the cells to stop translating –> want to implement a solution of things that will help the cell get over the issue
Global Regulation in Bacteria (Overall)
Overall - RelA is central regulator of tranlsation in bacteria
RelA = bacteria protein involved in stringent response (responds to cells that lack Amino Acids)
RelA = shuts off transcription and translation
Low Amino Acid Envirnments (Bacteria)
Low Amino acid envrimets = cells want to stop translating because there are not enough Amino Acids to make charged tRNA and make proteins
Signature of low Amino acids = more uncharged tRNA = tRNA is NOT bound to EF-TU –> signals that there is not enough Amino Acids
Signals that RelA responds to
- High amounts of uncharged tRNA (tRNA is not bound to Ef-Tu – not enough amino Acid around)
- Empty A site of the ribosomes because EF-Tu is not loading tRNA into A site
RelA Filling the empty A site
IN a race Actlayted tRNA loaded by Ef-TU will go faster BUT when A site is open if can be filled by a tRNA using RelA
- Normally tRNA is not free to react with RelA because they are often charged and bound to EF-Tu BUT when there is starvation they are free and cam bind to RelA
Process - When have empty A site and no charged tRNA –> reA bound to a tRNA wil fil the A site –> relA makes makes Magic Spot –> Binds to RNA polymerase and blocks intitaion for transcrtoion –> NO transcrtption meas have block for global translations
What does relA make
RelA makes a factor (factor = Magic spot) – pppGpp (5’ phos and 3’ phos on a guanosine) –> does this by putting together GTP or GDP together with phosphate from ATP -> get transcriptional respose (5’ phoshpate and 3’ Phosphate on Gauaine)
- (p)ppGpp = can’t initiation transcrtion because have phosphate on the 5’ end and 3’ end = blocks the 3’ OH = can’t get transcription
Magic spot = binds to RNA pol in bacteria and blocks initiation of transcription –> eventually leads to block in global translation
Mechanism for how RelA works
RelA reads the dual signal of binding to the emty A site and having many deactylated tRNA
RelA reacts with tRNA in solution –> RelA bound to tRNA binds to the open site of the ribosome –> RelA makes the magic spot
- When have freee tRNA and relA = get some magic BUT when have RelA bidning in the A site on the ribsome then makes a HUGE amount of magic spot (when have free tRNA and open A spot relA makes a lot of magic spot)
Cycle of RelA and tRNA binding
RelA binds to tRNA –> eventually the tRNA falls off and can get charged and end the cycle OR if can fall off and not get recharged and the cycle will restart with RelA
Global translation control in Eukaryotes (Overall)
Includes:
1. eIF2 phosphorylation (ISR)
2 . CAP dependent regulation (connections to TOR)
Initiating translation
Complexes used in intiation:
1. 4F complex – primes the mRNA
- 4E (within the 4F complex) binds to CAP
- 4F = prepares the mRNA for contact with the ribosome complex (ribosome complex will be bound to IF)
Process - Have Methionine tRNA in the P site that is reads to scan for the start codon –> interacts with eIF2 (eIF2 = GTPase) –> Methionine tRNA reads mRNA and hits an AUG –> Froms BP interaction with AUG –> when a codon and anticodon recognition occurs in the decoding center get GTP hydrolysis by EF-Tu
Inputs to activate Integrated Stress Response Pathway (ISR)
Overall - eIF2 gets phosphorylated –> leads get induction of stress response + get global shut down of tranlsation in cell
End - Get global shut down of tranlsation in cell BUT there are some genes that can escape repression
Pathway uses GCN2
GCN2
Kinase that functions in ISR pathway
- Similar to RelA
Responds to glucose deprivation, UV rdadtion, and Amino Acid
Yeast have 1 GCN2 kinase
Mammals have 3 related kinases
Discovery of GCN2
GCN2 was discovered by Alan Helanbrush –> plated yeast cells in the presence or absence of Amino acids –> Got a set of GCN genes
Mammal GCN2 Kinases
Mammals have 3 related kinases
- PERK – responds to ER stress = responds to misfolded proteins + Hypoxia + Oxygen-Glucose deprivation
- Misfoloded proteins trun on PERK –> get phoshphorylation of eIF2alpha
- PKR – responds to viral infections (responds to dsRNA)
- HRI – Heme response (Related to Iron deprivation + Responds to cytoplasmic proteins that are unfolded )
ALL GCN2 enzymes (GCN2 + PKR + PERK + HRI) respond to the same site eIF2a–> Phosphorylate SER 52 = get regulation
Regulation using eIF2a (overall)
GCN2 is activated –> phosphates eIF2alpha –> shuts down global translation
Normal eIF2a and eIF2b functions in transltion
Once GTP hydrolysis happens eIF2a needs GTP –> GDP exchange
- eiF2a gets new GTP by intercating with eIF2b
- eIF2b = Guanosine exchange factor
eIF2b binds to eIF2a bound to GDP –> exchanges GDP and GTP –> allows ongoing prtein synetshsis
How does Phosphorylation of eIF2a cause global shutdown
Overall - GCN2 Phosphorylated of eIF2a –> leads to trapping of eIF2b
When eIF2a is phosphorylated its affinity for eIF2b is high = eIF2b is sequester = can’t exchange GDP and GTP in cyclic manner
- LOW eIF2b levels when eIF2a is phosphorylated
- In general there is not a lot of eIF2b in the cell = don’t need to phosphorylate a lot of eIF2a to sequester all of the eIF2b
END result - eIF2a sequesters eIF2b = get transcriptional response and shut off global tranlsation because eIF2a can’t exchange GTP for GDP
Activating GCN2 (PAST thoughts)
PAST throughts:
GCN2 = recognizes tRNA (has a HIS RS domain – Histodytl tRNA syntehatase domain) –> HIS RS domain recognize and bind to tRNA = activates GCN2
- GCN2 domains include a kinda domain + a psuedokinase domain + HIS RS domain
New thoughts:
If have low Amino acids = low amounts of charged tRNA = teh A site of the ribsome may be emoty –> slow ribsomes –> GCN2 recognizes slow ribosomes
Things that Affect GCN2 activation
- Uncharged tRNA activates GCN2
- P stalk proteins on ribosome (collection of proteins moving around with long tails) –> P stalk proteins can actiavte GCN2
END – GCN2 is activated similarly to RelA
- Looking at tRNA (tRNA binds to the syntheses domain of GCN2) + looking at the state of ribsomes (if the arms are not occupied by recruiting EF-TU and tRNA THEN they tickle GCN2 and activate it
P stalk proteins
P stalk proteins (collection of proteins moving around with long tails) –> P stalk proteins can actiavte GCN2
P stalk porteins have long tails of the proteins that are positivley charged = act like a net to pull things in (Ex. Recruits EF-Tu)
Exception to GCN2 global shut down
GCN2 causes global reduction in translation BUT you might want to still make specific mRNA (NOW specific regulation NOT Global) - how do you make certain mRNA
Yeast - Found GCN4 gene is needed for survival under starvation
GCN4
GCN4 = Transcrtion factor – GCN4 goes to the nuclease and transcribes the right genes that respond to Amino Acid starvation (needed for survival under starvation conditions)
Don’t want to always make GCN4 (because when happy you don’t need GCN4) BUT want to have the gene present BUT not turned on so you can have a fast response
- Way to respond fast = if have mRNA there BUT it is not always being translated BUT it can be turned on during stress
How does GCN4 work
GCN4 = breaks the rule of using 1st AUG as the start site INSTEAD GCN4 = has 4 small Upstream ORFs
In a non-starved cell = have lots eIF2 + lots of translation –> ORF1 is translated –> ORF1 is short so not all of the initiation factors fall off = allows for second scanning mechasnism –> ribsome will translate ORF4 –> get termination and recycling –> NO EXPRESSION of GCN4 because translation stops at the end of ORF4
- ORF2 and ORF3 are not good start site sbecause they ahve poor Koazaks = not much happens when they are mutated (they are not very important)
- ORF 4 = good kozak –> has good termination site
When straving (want to express GCN4) have low eIF2 –> Make ORF1 BUT then scan over ORF4 (because it is not a GREAT start site) –> ribosome hits GCN4 (has good start site) –> get GCN4
- Weak signal of ORF4 is not enough when have less amino acids = get GCN4 in starvation
NOTE - System = not an on/off system have balance of strength of start sites
What happens to GCN4 expressiion if ORF4 AUG is mutated in media?
Answer - GCN4 epxression should go up
- In non-starvation conditions ORF 4 distracts the ribosome form getting to GCN4
What happens if the strength of the AUG start context of ORF 4 is increased under amino acid starvation conditions
Answeer - GCN4 expression goes down (because ORF4 will be more distracting
- Will get translation of ORF4 = will stop before GCN4 = no GCN4 expression
How do you study GCN4 system / test ribosomal profiling ise
To study – make a reproter and get readouts –> then do ribosomal profiling
Test ribosomal profiling using starvation (good model because know a lot about starvation)
- mRNA seq = don in all ribosomal profling experiment
Ribosomal profiling for GCN4 under amino acid starvation
Chart:
- mRNA seqeuncde in green – shows mRNA is there
- Bottom chart = starvation ; Top chart = rich media
In Rich media – see ORF1 being translated (have big peak of ribosomes in ORF1) + Less ribsomes in ORF2 (less tranlsation) + have a small amount of ribsomes in ORF3 + Few ribsomes in GCN4
- Rich media = not tranlsating GCN4
IN starvation – Have more ribosomes in GCN4 (Means GCN4 is expressed and being translated)
- ALSO Have many ribosomes before ORF1 (unexpected)
New expeirment based on fact that there were ribsomes BEFORE ORF1 (test the starvation model post ribosomal profiing)
Looked at Upstream of the ORFs for near cognates of AUG that might result in ribosomes starting there –> THEY found non-conical start sites such as UUG and AUA upstram of the ORF = maybe those are start site = have ribosomes upstream of ORF
After looking for conical starts sits – Made reporters for GCN4 expression (linked to lac z) where they mutated 1 of the non-conical starts sites or the other non-conical start sites or both of the non-conical start sites –> Asks how that affects regulation
Results:
Two bars for each condition in chart – Untreated and Starved
- WT = no GCN4 in rich media ; have GCN4 in starvation
- When Muatant of 1 uORF + Mutant of other uORF + mutant both you maintain regulation
END - Don’t know why have ribosomal reads upstream of ORF1 BUT the reads don’t seem to be important for the regulation
Mammalian Global Regulation
Uses ATF4 (similar to GCN4)
Mammals = have 4 different eIF2a kinases –> affects eIF2 association with eIF2b
ATF4 = Transcrtion factor
ATF4 = has two upstream ORF
- ORF1 = always translated
- ORF2 = overlaps with ATF4 (when make ORF2 then NOT making ATF4) ; ORF2 in ATF4 = similar to ORF4 in GCN2
Normal = translate ORF 1 and ORF2
Starving = no eIF2a = make ORF1 BUT fail to make ORF2 –> get ATF4 –> ATF4 is a transcription factor that goes to the nucleus and turns on genes to save the cell from whichever stress it was
Integrated stress response
Integrated stress response refers to the shut down of translation AND a transcription response that is needed for cell survival
The integrated stress response is a major mechanism of translational control downstream of four related kinases (GCN2, PERK, PKR and HRI)
What is needed at the end of the Intergared Stress Reponse Pathway
At the end = need to turn off the stress signal at some point
- IF ISR is not turned off = leads to apoptosis = needs to be regulated
Solution – have phosphatases that turn off the phosphorylation
Prevelence of uORFs
Many genes have uORFs and don’t use 95% rule of kozak (dones’t use first AUG of the main instad uses AUG in uORFs)
- uORFs are regulatory (know they are regulatory because mutate the upstream AUG and see if make more of downsream gene) = central to gene specific regulation
Yin and Yang of ISR
Chart shows the Level of phosphorylated eIF2a vs. Level of translation
Overall:
High phosphorylated eIF2a = low translation
Low Phosphorylated eIF2a = have high translation
Have zone in middle where fix things
Importance of ATF4 pathway
ATF4 is such an important pathway that companies tried to make drugs that target the pathway –> did a drug screen
How do you do a drug screen - Use reporter + cell line (NOT done in mice) –> need readout
- Make a reporter with ATF4 (ATF4 is hooked up to firefly Leusiferase –> can look at cell to see if have leciferase)
- In reporter whether or not you have luecerferase is a function of whether or not uORF1 or uORF2 blocks translation of ATF4
- Reporter is looking at expression of ATF4
Thapsagardin
Promotes the misfoloding of proteins in ER = actiavtes the ATF4 pathway through PERK
Thapsagardin = activates PERK kinase –> PERK phosphorylates eIF2a –> Phosphorylation allows AFT4 to be expressed (because ribosomes can ignore ORF2)
No Thamsargardin = not making ATF4
Have Thapsagardin = make a lot of ATF4
Shows reporter works
Doing drug screen
- Once show reporter works–> Put cels in wells in dish and add durgs to each well and ask if there us anything that blocks production of ATF4
Chart - Thapsagardin signal – Want inhibition of ATF4 even when add Thapsagardin
- Found 400 drugs that passed some threshold –> then narrowed to 28 –> then chose ISRIB
- Once found ISRIB drug = need to titrate and show the binding constant (found trans vs. Cis compounds have different binding constants)
ISRIB (overall)
Drug that blocks expression of ATF4 in cells that are exposed to Thapsagardin
How does ISRIB work: ISRIB binds to eIF2b
Why is ISRIB so good
Because ISRIB binds to eIF2b = functions at the bottom of the pathway
EIF2b = guanosine exchange factrs –> means ISRIB functions at the bottom of the pathway (means it should be effective on every feed in pathway (should work on PERK and HIR etc) ; should work for all GCN proteins)
ISRIB titration
ISRIB woks in certain titration range and then truns off
EIF2b is active as a tetrome and less active as an octomer –> ISBRIB = activates the tetromeric form of eIF2b and inhibits the octomer
ISRIB = Function like a Reohstat for eIF2b activity
- Low amounts staples together eIF2b and enhances activity
- Too much stabilizes tetramer rather than the octomer
Ways to target Integrated Stress response Pathway
There are multiple ways to target the ISR (effects on health are not always predictable)
- Many companies want to target this pathway
- Pathway is an example of how to globally shut down translation and specifically express genes
Example to target ISR = ISRIB (targets the last step of ISR)
4E and 4E binding proteins
4E binding proteins bind to 4E –> regulate 4E (when 4E binding proteins bind to 4E they block 4E from recognizing CAP)
4E binding proteins are regulated by phosphorylation –> Phosphorylate 4E and 4E binding proteins = 4E and 4E bidning proteins can’t intercat with one another + Phosphorylation of 4E allows 4E to bind to CAP = get tranlsation
- Phosphorylation of 4E and 4E binding proteins promotes translation
How is TOR regulated/ the consequences for TOR regulation
Western blot – shows phosphorylation states for TOR targets
- Treatment groups = Vehicle (normal cells) + Rapa (inhibits TOR) + Torin (inhibits TOR) + No amino acid group
- eIF2a – has two bands (top is the phoporylated state ; bottom is the protein) –> shows the eIF2a doesn’t talk to TOR (because no change in any of the treatment groups)
- S6Kinase and 4EBP = major target of TOR –> in all three conditions (inhibiting TOR and AA starvation) = have a decrease in amount of phosyrlation of 4EBP + S6Kinase (Phosrylated in vechile and then blcok phosphorlyation in the inhibitors/AA starved)
Polysome profile and TOR inhibition
Healthy cells = have a lot of polysomes on mRNA
When add TOR inhibitor = number of polysomes decreases and 80S increases = have a block in tranlsation
- Have global shut down in tranlation when turn off TOR or starved for amino acids
Ribsome profile and TOR inhibition
Looking to see if yo turn off all of the genes or if there are some that are still expressed (what mRNA are still bound to ribosome ween have Torin or no Torin)
- Looks at translation efficiency –> asking how effectively mRNA is being translated –> SEE some mRNA are better translated and some are less well translated
Results:
1. GCN4 = expressed in TOR inhibition (in the blue on the chart)
- Blue = GCN4 – gene you make in crisis
2. Some genes are not tranlsated
- Green = genes that you stop making (mRNA of genes that are not tranlsated under the conditions
Gene Seqeunces of TOPs
Different websites look at different start sites
- Found 100 mTNA in TOPs that are well regulated in known TOPs they already knew about
- See that some websites didn’t calls something a TOP or they called it a TOP like mRNA (disrupted TOP) –> sows that make sure you look at the TOPs (how you define them is importnat)
- When get a list of genes that are well regulated – look if they are TOPS or to and why they were defined as TOPs and are there different ways to define TOPs or where the Transcription starts sites is
Results when sequence each fraction in Ribosome profile
Left charts:
See Actin in deep fraction (not in light faction) (fraction 6/7) ; actin is an example with high translation (lots of polysomes)
Right Chart:
DMSO Treated -
1. DMSO A260 is well translated (lloks the same as active)
2. ALL the non-TOP mrNA (chart at the bottom) = all in the deep fraction (look like actin) = well tranlsated
3. TOP gene - Every TOP is half loaded with ribosomes and half not loaded (averge Ribosomal profile would not be able to show the biphasic natre)
Add Torin (inhibit TOR) - get global movement in if the mRNA is translated
1. In the non-TOP with TOR (bottom right chart) = see shift in many mRNA = have global movement in whether or not the mRNA is being translated
2. In TOP gene = fully turn off the TOPs (TOPs are specifically truned off)
Inputs into the CAP recognition complex
There are multiple inputs into the CAP recognition complex
TOR = feeds to S6K + Feeds to 4E Binding proteins (when not 4E avaible) + Feeds to MINK1/2 (phosphorylates 4E binding proteins)
SHOWS that many signaling pathways are important for cell growth – signals feed at the cell membrane and mTOR is a major regulatory node
Virus Transcription and translation
Viruses – make their own mRNA BUT doesn’t want to compete with the Eukryotic system because need the host cell to survive
Viruses will cleave PolA binding protein + cleave 4G complex + decaps host mRNA –> ALL disrupts the elements for CAP dependent translation –> once CAP dependent mechansim is detsroyed cells/viruses still need to have a way to translate important things for host cell to survive
Solution = Iris –> iris = dones’t need CAP – just needs ribsomes and tRNA and syntehtases
How do you look for Iris Function
How to look for Iris function – use bicystronic reporter assay
Bicytronic reporter has 2 different reproters (Example Renella and Firefly – have different colors so you can measure both indepentely
How does bicytronic work:
- Normal cell – scanning happens = reacignize the first ORF BUT should not recgiizie the 2nd ORF (Because the first ORF would lead to normal termination and recycling and would not get tranlation of the second ORF)
- In Bicyrtonic - Put in an iris seqeunce ) to see if can get expression of the second reporter –> if get expression from second ORF then there must something sepcial about the sequence that does NOT depend on CAP
How does Cricket work
Overall - Cricket places the Ala codon in the A site of ribosome –> doesn’t need to recgizne CAP or eiF2a to load initiator tRNA
- Doesn’t need Met tRNA because Cricket has secondary structure that looks like tRNA/mRNA decoding event (looks liek start site BUT it is not start site it is a peice of mRNA)
How does this happen - Cricket will bind to the P site using tRNA like structure then Ala can come in –> translation can start
Riboswitches
Riboswitches = small elements in 5’ UTR of bacterial genes
- Riboswitches = fold to a 3D structure in cis to gene of interest
Ex. can bind thymine pyrophsphate –> block the thymine pyrophasphate gene from being expressed
- Lots of thymine pyrophasophate = don’t need to make the gene that makes thymine pyrophate (auto feedback loop)
What happens:
1. High Thymine - thymine binds = SD is sequestered in some RNA secondary structure
2. Low Thymine - the riboswitch structure is different = SD is available = get translation
Gene specific regulation (Euk)
Can be done through:
1. 5’ UTR binding porteins
2. 3’ UTR elements that regulate intiation through CAP dependent mechansims (MORE common)
- 3’ UTR regulation = very common (very common during development)
Example of 3’ UTR mediation regulation (in C. elegans)
Have different proteins and mRNA in the worm development
Example (image) - oocytes are moving down a track and are released for fertilization –> Can see that there are different patterns of where the proteins are found
Experiment to show proteins are not regulated at the level of transcription
To show that the proteins are not regulated by transcription – put the genes under a constitutive promoter + put GFP fused on the 3’ UTR of the the different mRNA –> ask where GFP is expressed
- Constitutive promoter means the protein would be made everywhere/all of the time)
Results – show that GFP expression a the begining or the middle or the end in a manner that depends on which 3’ UTR they have (shows 3’ UTR drives protein expression in a localized manner)
Specific example of 3’ UTR regulation
miRNAs binds to the 3’ UTR for regulation (common regulating feature in mammalian genes)
- There are 100s of miRNA and each miRNA has multiple targets = many genes are regulated by miRNAs
- miRNA is not turning genes fully on/off more like changes regulation by 20%
miRNA = Usually binds to the 3’ UTR- miRNA binds to 5’ seed sequences in context of AGO protein –> targets the mRNA for degredation
How does the protein on the 3’UTR talk to the 5’ end
Overall - based on whether or not 4E is compenent and can recruit 4G
- Shows how feature in 3’UTR can regulate specific mRNA rather than the whole cell
How does this work - In the tranlsatonally inactive mRNA AAUAAA that is normally recognized by CPSF in the nuclues is not recognized + 4E is bound to CAP BUT 4E is not bound to 4G instead 4E is bound to Maskin (Maskin is bound to CPEB) CPEB binds to the CPE element in the 3’ UTR
- Maskin has 4G like element
THEN a kinase becomes active when sperm enters teh cell = get phosphyration = phosphrylatiion leads to the recuritemnt if CPSF that will clevae AUAAAA seq –> get PAP -> PAP puts on the polyA tail -> PolyA binding proteins is recruied (recuites eiF4G–> eIF4G have affinity to displace the maskin
- Example of a sequence in 3’ UTR that does what Torin did genrally (Torin played with how likley will you make active complex based on 4E binding proteins that sequester the protein)
How does the cell know that the stop codon is any different that other stop codon
Overall - Cells knows based on the fact that stop codons are almost always in the terminal exon because of NMD + based on the fact that during splicing the splicing machinery makes an EJC
- By contrast the start codon can be in any exon
Process:
Normally - Normal stop codon = clear all EJC then hit Stop codn (no EJC downstream)
When have premature stop codon - ribosome sees a stop codon upstream of the EJC they know is is a bad mRNA = recruits UPFs (UPFs = proteins that are important for nonsense mediaed decay) –> degraded the mRNA
