Lecture #10 (Translation) Flashcards
Central Dogma
DNA –> RNA –> Protein
Process by which genetic information is transformed into the proteins in the cell that preform functions of the cell
What is regulation
Regulation is often just changes in thermodynamics and kinetics of core steps
- Regulation usually doesn’t create a whole new way of doing things
- Regulation is driven by rate constants and binding constants
- Don’t need a whole new system to chnage expression INSTEAD can have a small chnage in 1 factor that can affect the output
- On/Off regulation can happen BUT having a smaller change (ex. A 2 fold chnage) can still be relevant (2 fold chnage can have a big cumulative effect)
Story about not having a whole new way of doing things
At discovery of RNAi people could kncokdown genes and look at gene function
A researcher was looking at alternative splicing and found all of the hits in the knockdown were for core splicing factors
- The guy that discovered this was suprised – he thought he would get a whole new set of factors BUT he got the same splocing factors they already knew existed
SHOWS it is not whole new systems being created
Where is most gene regulation done
MOST of biology = driven by transcriptonal regulation
- Transcrtional regulation = use Transcriptino factors that affect which transcripts are being made
Where can you regulate gene expression in translation
- Initiation
- Elongation
- Regulation during Termination/recycling (Used for quality control/mRNA surveillance)
- Quality control is critical for recognizing the termination complex
Regulation during Intiation
(Have core translation and initiation factors + Have Upstream translation and alternative start sites)
- MOST of regulation = in initiation (decide whether to actually translate)
Regulation during Elongation
Less common but happens
Can include elongation speed + ribosome pausing + Cna have co-translational folding and localization
Example - Secreted proteins can cause elongation to stal before finding the ER
Bacteria mRNA structure
Bacteria mRNA= polycystronic –> have multiple genes on 1 mRNA
Bacteria = can’t have ribsome start ONLY at the initial 1st AUG of the first gene because at the end of protein synthesis you dissocate the ribsomes = you would dissociate the risbosomes at the first stop = would no get translation of the next gene
- Bacteria – tranlsation is NOT a processive mark
Solution - Each ORF has its own AUG + own stop codon + own Shine delgarno region
Solving issue with Bacteria mRNA structure
Bacteria have an indepenet internal intiation site –> can start translation at each indivual AUG for each gene using the Shine delgarno Sequence
Shine Delgarno Sequence
Shine delgarno = polypurine stretch upstream of the AUG start site
- Shine delgarno sequnece use for regulation of protein synthesis
Function - facilitates the identification of the translation start site (helps ribosome find the AUG start site)
Experiment to find Shine delgarno sequence – saw ribsomes looked different in different bacteria AND the shine delgarno sequence looked different in different bacteria
- Ribosomes and SD had co-varaiton in patterns (SD and ribosomes matched each other)
Eukaryotic mRNA Structure
Eukryotic mRNA = monocytrinic (only codes for 1 gene)
- mRNA has 1 ORF + has 1 AUG + 1 stop codon
- Allows eukaryotic ribosomes to use scanning
- mRNA also has polyA tail + Eukaryotic mRNA has a 5’CAP (CAP is recognized by eIF40)
Translation (overall)
Translation = proccessive mark along linear template
Along template have adapter molecules (tRNA) attaches to Amino acids building blocks –> amino acids are put together to make protein chain
Genetic codes
Dictates how get form nucleotides to amino acids
Have 4 Nucletides and 20 AA –> Code needs to be a 3 letter code to coevr 20 AA
- 4^3 = 64 different letter groups that can specify the 20 AA (have 64 codons)
Genetic Code is redundant + Non-ambgous
- Redundant because Many AA are specified by >1 codon
- Non-ambiguous = all codons code for something
Aminoacyls –tRNA
Adapters – interprets genetic information and brings amino acid
- tRNA 2D structure = Clover leaf ; 3D structure = L shape structure
- Bottom = have anticodon with 3 nucleotide motif that interacts with codon in mRNA
- Top = Acceptor end of tRNA (3’ terminal CCA tail – where AA is placed)
Are there 61 tRNA in a cell
Never 61 tRNA in any given cell – often have 30-40 tRNA in cell –> means certain tRNA recognize more than 1 codon
1 tRNA can recognize more than 1 codon because of the wobble position –> 3rd nucleotide in codon can be different and 1 tRNA can still recognize even if there is a mismatch
How do 20 Amino acids get coupled with the tRNA
Uses Aminoacyl tRNA synthetase
Ribsomes
Has 2 SU:
1. Small SU = interprets the genetic code (has mRNA/tRNA paring)
2. Large SU = where peptide formation takes place
Most of ribosome = made up of RNA (2/3 of mass is RNA ; 1/3 mass is proteins)
Core initiation factors
Core initiation factors – guide initiator tRNA to P site + Subunit joining
Bacteria = IF1,2,3
Euk = eIF1A, eIF5B, eIF1
- eIF2, eIF4E, eIF4G, eiF4A, eIF4B, eIF3, eiF5 (Bind to CAP and PolyA tail + facilitate scanning )
IF1 = eIF1A
Factors for elongation
Bacteria - EF-Tu, EF-G, EF-T
Euk - EEF1a, eEF2, eEF-1B
Factors for terminiation and recylcing
Termination:
Bacteria – RF1, RF2, RF3
Euk – eRf1, eRF3
Reycling:
Bacteria – RRF, EFG
Eukryotes – eRF1, eRF3, ABCE1
What are most enzymes in protein translation
MOST enzymes in protein translation = GTPase enzymes
Translation Initiation (overall)
Process by which an initiator tRNA (always methionine) find AUG start site and the ribosome SU assembled on the start codon
IF1 and IF3 vs. EIF1A and eIF1 = bind in places where they don’t want tRNA to bind
- IF 3 binds to small SU ; IF1 binds on the other side –> place where we want initiator tRNA to bind is between IF1 and IF3 = block the tRNA form binding from the wrong tRNA binding site
Bacterial Initiation
Overall - Uses the Shine Delgarno Sequence
- NOTE - Large SU does NOT play a role in initiation
Process:
1. Small SU binds IF1 and IF3 (IF3 is in the E site and IF1 is in the A site)
- Binding of IF1 and iF3 ensures that only the P site is open (P site is where intiation takes place)
2. Small SU (now bound to IF1 and IF3) will bind to the mRNA
- Small Su binding to the mRNA - Bacterial Small SU rRNA has anti shine-delagaro motif that is compelnetary to SD motif - binds to it = tethers mRNA to the small SU of ribsome –> AUG is position in the P site of the ribosome where tRNA can find it
3. Once small SU is bound to mRNA –> Methionin tRNA will find AUG start site in the P site
- Finds AUG start site through sequnece cues (include the SD sequence)
4. Once the Met tRNA binds in the P starts site –> IF1 and IF3 go away
5. Once IF1 and IF3 leave IF2 (GTPase enzyme) comes in and catylyzes the joining of the large SU on the ribsome = elongation can start
Eukryotic Tranlsation Initiation
Eukaryotic = use scanning –> look for the first AUG from the 5’ end to be the start site
Process:
1. Scanning occurs with initiation factors –> forms a circular complex
2. PIC will bind to the 4F complex (PIC includes 1, 2, 5, 1A, 3 + Small SU)
- eIF1 and eIF1A = binds to the A and E sites in the ribosome
- tRNA binds to the ribsome using eIF2
3. EIF2 = binds to the inteer tRNA –> joins the CAP complex and sans the mRNA looking for the compleet of AUG –> once finds teh complement it will engage –> once enages it will do GTP hydrolysis
4. Large Su will be added using EIF5B (eIF5B reciginzes AUG = joins the large SU –> THEN intiation factors leave)
- IF2 = like EIF5B –> both help the large SU join
Role of the Eukryotc Initiation Factors
Intiation factors = 4F complex – inlcudes 4G, 4E, 4A, and 4B
Function of the 4F complex = bind to CAP to prepare mRNA
- 4G = binds to the polyA tail –> > form circular complex
- Prepares the dsRNA to encounter a ribosome
eIF2
eIF2 (GTPase) = carries the intiatior tRNA
- There is a lot of regulation using eIF2
Kozak Sequence
Eukaryotes = have Kozak Sequence –> AUG tend to be surdouned by certain nucleotides that help AUG be a better start site
Ribsome does NOT base pair with teh kozak sequence
Oris = more like shine delgarno seqeunce in Eukryotes
Elongation Steps (overal)
- Selection of correct tRNA (Decoding)
- Peptide bond formation – AA are ligated to one anotehr
- Translocation– whole complex is moved along mRNA template to open A site for next tRNA
Elongation Decoding
Overall - aminoacyl tRNA goes to the A site in a manner that matches the codon
Uses ETFU (bacteria) eEF1A (Eukryotes)
- EFTU = binds to the aminoacyl tRNA –> Helps load tRNA quickly and with high fidelity to active site of ribosome (in decoding center)
EFTU loading of the tRNA
EFTu loads tRNA into ribsome using GTP hydrolysis
- Once tRNA is loaded there are two opportunities where ribooeme can reject the wrong RNA = facilitated by EFTU
Have nucletides in rRNA in SU in decoding center where codon anticodon interaction is interpreted –> movement of Adenosine nucleotide to a new confirmation is nececasery for ribosome knowing this is a good tRNA to keep
Elingation - Peptide bond formation
Peptide goes form tRNA on P site to tRNA on the A site
Location – Occurs in RNA rich active site in large SU of ribosome
Process - Guanosine binds to CCA on tRNA –> puts tRNA in active site
Elongation Reaction (How does catalyisis Occur)
Reaction - Attack of nucleophile on Electron deficient bond
Ribosome facilities chemistry by bringing compoenent together with conserved elements (A loop and P loop use W/C pairing interactions with CC of 1 side and CC of the other side to bring the two substrates together to do nucleophilic attack
Elongation (Transloation)
After peptide bond formation tRNA is in a hybrid state –> resolved by translocating factors
tRNA and mRNA need to translocate –> mRNA/tRNA complex is moved to open A site to be open for new tRNA to come in
EFG (GTP Hydrolaze) = does the translocation
- GTP hydrolysis coupled to movement of complex
- Domain 4 of EFG = binds in A site of ribosome –> promotes the foward movement of the mRN/tRNA complex by binding in site and displacing the tRNA that was there before
tRNA binding sites in ribosome
- P site (peptidyle site) - Has the growing peptide chain
- A Site (Aminoacyl site) - where trNA are loaded by EFTU
- E site (Exit site) - where tRNA sits on the way out
Tranlsation Termination
Overall - Stop codons need to be recognized by termination factors
- Termination factors - Have end that recognizes the codon + end that promotes hydrolysis of peptide chain so it can leave
- Termination factor structure = similar to tRNA structure
- Termination factor = binds in the A site to recognize the stop codon
Translation recycling
After termination - Ribosomes Complex is broken apart to allow for next round of translation
- Bacteria and Eukaryotes use different factors
- Uses ATP
Bacterial Vs. Eukaryotic Termination Factors
Bacterial vs. Eukaryotic termination factors are NOT related
- Means termination evoloved twice
Active site of the termination factor in BOTH Eukaryotic and bacteria = GGQ (Uses water for catalysis) –> BOTH bacteria and eukaryotes came up with the same solution for catalysis
Polysomes
Translation = takes place on polysomes
Polysomes = 1 ribosome that intiates and moves down AND then another ribosome will initiate behind it (mRNA is coated in ribosome complexes)
In Vivo translation
Translation happens in continuous process in cell = have mRNA template in cell where ribosomes are loading -> translated until end –> release peptide BUT cell doesn’t wait for release of peptide until it starts again = in cell it is really a polysome (multiple ribosome on 1 mRNA continually making peptide)
Image – shows multiple ribosomes laoding on a long mRNA
Tool to study translation
Polysomal profiling
Experiment:
1. Take cytoplasm extract from cell
2. Place cytoplasm extract of a cell in tube that has a sucrose gradient
3. Spine the tube to get a sedimentation gradient
- Ribosomes will go to the bottom of the tube (Split into different factions in gradient along tube –> then fraction with 30S ribosomes –> then fraction with 50S ribosomes etc.)
4. Read the ribosomes uses absorbance 253
- Absorbance 250 = reads RNA (ribsomes are made up of RNA)
NOTE - Don’t know anything about mRNA amount JUST how many ribosomes are on mRNA
Chart from Polysomal profiling
Overall - shows how many ribosomes are on RNA transcripts
Peak = the fraction that will contain that type of ribsome –> fraction that has all of the a 30S SU or fraction that has all of teh 50 S SU
- 30S and 50S can be free ; fractions after will be bound to mRNA (show the amount of transcripts that have THAT amount of polysomes)
- Before peaks = free ribosomes –> some of the 30S and 50S are free (not engaged with mRNA)
- Peaks towards the right = polysomes on mRNA (KNOW they are attached to the mRNA because the only way for them to migrate in the sedimentation gradient is if they are attached to mRNA)
Ribosomes SU in bacteria vs. Eukaryotes
30S, 50S, 70S = bacteria
40S , 60S, 80S = Eukaryotes
Experiment using Polysome profiling (normal conditions vs. Hypoxic conditions)
Chart is polysome profiles of cells in normal conditions or hypoxic conditions
- Shows hypoxic cells = little translation (have 80S BUT no polysomes)
How do you know the what mRNA the ribosomes are on and how well they are being translated
Run a northern blot (looks at RNA)
Northern helps find RNA of interest out of 1000s of RNAs by using a radioactive probe that hybridizes to RNA of interest
Experiment using Polysome profiling (normal conditions vs. Hypoxic conditions) + Northern Blot
Normal condition – ALL mRNA are well translated (have high polysomal fractions expression)
- In northern - RPLS has expression in polysome and fraction 4 (seen in 2 places)
Hypoxic - HIF1 alpah is there BUT GAPDH is less and RPL is shut off
Shows genes that are no longer translated when cells don’t have enough O2 BUT HIF1 is still there (HIF 1 = needed when cells are under stress)
Con with qRT-PCR
Fragments amplified are 2-300 BP BUT mRNA transcript is bigger (only looking at a part of the transcript and assuming that reflects the whole transcript)
Polysomal Profiling Example #2
A – shows a normal tranlating cell
B – Only has 80S particle with some 40S and 60S
C – Only has 40S and 60S
How do you make a cell go from normal in A to only have 80S,40S, and 60S in B? –> cut the mRNA with RNAse (Have no RNA to elongate)
- Add RNAse = only get 80S
How to get C = add EDTA –> causes the ribsomes to dissciate = only get 40S and 60S fractions
- EDTA works because ribsomes need Mg
Purple trace is regulated and the blue line is unregulated (grey is actin) - What mechanism is at place?
Principal = following where mRNA goes (percent mRNA in each fraction)
- Exp Set up - Syn has miRNA biding sites that regulate SYN mRNA ; FLP = mutations of the miRNA binding sites
Based on chart = know it is an initiation block –> because not getting 80S ribosome (mRNA is not getting loaded with 80S ribosome because have dip at fraction 4 which would have 80S)
- ALSO when have the mutation = there is no polysomes = issue with initiation
What do different blocks look like on polysomal profiling?
IF had elongation block = the peak would be shifted to the right (would have loading of ribosomes)
- Ribosomes would get stuck in ORF and would get higher % mRNA 80S fragment
IF have termination block = would be shifted deeper to the right (would have polysomes)
Tool to look at ribosomes #2
Ribosomal Profiling (Use high throughput sequencing) - Shows where the ribosomes are + which mRNA the ribosomes are on
- Can be done on single gene or multi gene
Process:
1. Add cyclohexaminde = inhibits elongation (stops translation)
2. lyse the cells to get the polysomes out of the cells
3. Use nuclease to generate monosomes
- Will have some RNA that was protected by the ribosomes
4. Purify the ribsome protected fragments
5. Add linkers to the fragments
6. Get reads using NGS
7. Align reads to transciptome and see where they are
Ribosomal Profiling data
Overal - shows where on the mRNA is the ribosome bound
Higher peak = higher percent of transcripts with ribosome at that location
Chart:
Y axis = how many ribosomes in that region
- How may reads for that ORF relative to the total number that you have sequenced–> percent of reads that had a ribsome
X Axis - Shows place in the genome
Chart shows few ribsomes on 5’UTR + most ribosomes are on the ORF
- Ribsomes on 5’ UTR = scanning
Higher peaks on Ribosomal profiling data
Peaks = indicates that the ribsomes are slower at that location (have more ribosomes stuck there) ; lower peaks = ribosomes go faster over that location
How do you form most conclusions
MOST conclusion = not looking at 1 specific mRNA (not 1 gene) INSTEAD use a meta gene analysis
Length of most ribosome protected regions when using Cylcohexamide
Chart - shows read size (distribution of the footprint size) –> shows that the ribosome protected regions are 28 nucletodes long
Meta Gene analysis
Take all of the reads (that are 28 nucleotides long) –> Align to the transcriptome
Looking at 1 gene doesn’t tell you as much –> look at meta gene instead (MANY genes) –> take 100 milion sequence reads –> identify all Ribosome protect regions at the start codon —> Align all of the start codons together
Chart - Peaks = ribosome density
- See few ribosomes in the 5’ UTR (few reads map to the 5’ UTR)
- Have peak at the AUG start site (high density of ribosome) THEN have peaks as move out from start site
Trend of peaks in Meta gene analysis
Peaks show periodicity of 3 BP (ribsome is moving 3 nucleotides at a time along the mRNA in the cell)
- Can see when looking at many genes –> know it is genome wide
Speed of intiation and termination based on chart
Based on chart - recognition of the start site is slow because have more ribsomes there (they are stuck sitting there longer)
- Accumilation of ribsomes = suggest they stay longer there = slower
In a separate experiment can align all of the reads to the stop codon (before it was the start codon) - see that stop is also slower than elongation
Assessing to see if Polysomal profiling works
Goal - Show that ribosomal profiling predicts the amounts of protein being made (Better method than using mRNA)
Left Charts: Looking to see if the mRNA correlates with the protein amounts (IF correlated then have 1,1 on chart) –> Shows that mRNA does not correlate well with protein amounts
Y Axis = Have protein abudnece
X Axis = mRNA
Each dit is a proein in the cell
Right chart: See that there is a better correlation –> Shows ribosomal profiling is a better reflection of protein output
Y Axis = Have protein abundance
X Axis – Ribosome density (ribosomes on a given mRNA)
Ribosome profiling in E.coli Stoichiometries of protein complexes
Correlated ribosomal profiling data with known abundance of protein complexes
Chart A – shows the operon with 9 genes – cell needs to know how much of each gene to make
- Know there is a 1 B made : 10 E made
- FOUND that the stoichemtry was reflected in the amount if ribosome reads on the ORF ( Higher ribsomal density when need 10 copies of E ; lower ribsomal density whne have 1 copy of B)
- Have a correlation between the stoichemtry of the complex and the amount of ribsome reads on the mRNA of that region (amount of mRNA that was protected by the ribsomes)
Chart B – shows that this concep applis to multiple complexes because ribosomal reads fall on the diagonal line
Ribosomes + Stoichemetry of complexes
Ribosomes translate the right amount of proteins to make protein complexes in the right stochemtry
Protein Synthesis rates and copy number
Chart - shows the synthesis rate correlates with the known copy number in the cell (how many copies of something you need) ;
- More copies = have higher syntehsis rate
What is happening on a single gene
When have single ORF – see low ribsome density in the begining (have fast elongation) and high ribosome density at the end (slow elongation)
Means there are sequences that are heard to tranlsate at the end (ex. Arginine codons that are harder to translate)
‘Texture’ on a single gene can tell you about the specfics of elongaton
Issue with ribosomal profiling
Issue with profiling 1 gene - ribosomal profiling data on any 1 gene = high ribsomes on transcrtpt may or may not mean high out (if robsomes are not mving then won’t get any protein)
- Ribsomal profling = stead state experiment – don’t know if ribsomes are moving
Profiling data reflects anticipated stalling
Charts show - what codons the ribosomes are on –> when open the cell the ribsomes should be sitting on the codons in specific amounts
- REALLY THESE charts show that the
ribosomes are not getting stuck as much as they should when only add CHX
Experiment 1 = took yeast samples –> add gamma toxin to some
- Gamma toxin = protein that cleaves the glutamate tRNA in cell = depletes number of glutamate tRNA –> expect that the ribosomes would get stuck on the glutamate codon (GAA) because there is not enough tRNA
Chart:
Ribosomes density on the different codons in Gamma toxin treated vs. Untreated –> See that when adding Gamma toxin have more ribosomes stuck on GAA
- When only add cyloxadine – there is no difference in treated vs untreated
- When add gama – have higher ribosomal density at GAA = ribosomes are stuck
Experiment 2 – remove Histadine = ribsomes should get stuck on the histadine codons
- Chart – the ribosomes are a little slower ve the histadine codons (CAU and CAC are above the line) but not really reflecting how slow i should be
Improvements in Ribosomal profiling
Chart:
CHX = gets grey peak at 28 nucletide footprint (get reads that are 28 nt long)
Use CHX/TIG or CHX/ANS (different elongation inhibiotor) = get peak of reads 28 nucleotides long + get reads hat are 21 nucleotides longer –> MEANS that TIG targets a different step in elongation
- 28 nucleotide fragemnts = due to translaotion inhibiter (CHX) or Accomindation (TIG)
- CHX/TIG = better at capturing ribsomes + gives 2 footprint sizes
What do the different elongation inhibitors inhibit
CHX = blocks translocation
TIG = Blocks bidning of tRNA
ANS = blocks peptidly transfer
Original expeirmnts didn’t work as well because when use CHX to block translocation the ribsomes are still runnning = still moving in lysate = move to the 28 fragment –> gets stuck at the 28 footprint then accumilate = all reads were 28 nt long BUT when add TIG you trap ribosomes earlier = get 21 nt
Prolfing data that DOES reflect anticpated stalling
When sue CHX = don’t get stalling at GAA BUT when use CHX/TIG (double stalling) = NOW see stalling at GAA
- Ribosomes are slow over the GAA codon when TIG is present ;
- When TIG is present HIS is slow in the prescence of durg that blocks histaine
- data = meta analysis ; 1 gene can’t tel you codon level information
Means that you can capture exactly whee on a given transcript the ribsome is
WHEN ONLY HAVE CHX YOU DON’T SEE THE EXPECTED TREND BECAUSE NOT SLOW AT GAA BUT WHEN ADD 2 INHIBITORS YOU DO SEE THE EXPECTED TREN
Typical pausing during elongation (Single gene anlysis)
Overall - Looking to see if there is a problematic sequence
Looking at WT vs. Mutant E.coli (mutant lack a factor) - Want to know if you deplete 1 factor (delataEFP) - see huge increase at 1 site increase in ribosomal density?
Shows - High pile of ribosomes on Polyproline (PPP) motif = cell struggles with tranlsating prolines
- See 1 gene is very ‘bouncy’ (uneven ribosomal density) - not looking at ‘bounce’ at any 1 gene
Means that the factor not found in mutated protein is used to get ribsomes passed iterative prolines because the mutated that lacks the factor stuggles with prolines
Typical pausing during elongation (Meta gene analysis)
Looking at many reads (look at all reads with single prolines ; all reads with double proline s; all reads with triple prolines) and ask if proline is generally problematic
Shows - 1 proline = not an issue (don’t get buid up of ribosomes) ; 2 prolines is a small issue (have more ribsomes there) ; 3 prolines in a row is very probalamatic (have big build up of ribosomes)
- Bigger peak = more problematic – ribosomes have a harder time getting through it
Example #2 of site specific pausing
Looking at 21 nt fragments in WT stressed vs. 21 nt fragments in WT unstressed–> See 61 dots for all codons that are not stop codons
Shows 4 problematic codons (CCA, CCU, CCC, CCG – ALL proline codons) –> tels you than in stressed cells here is an issue translating proline
- Have ribosomes on these codons more often in the Oxidative than in untreated
Meta anlysis chat - align the 100 million reads of different genes at a given proline codon
- In the untressed cells the ribsomes are a little slower over the proine BUT in streassed cells the ribsomes are much slower over the proline (have huge peak)
SIngle gene vs. Multi gene
Power comes from multi gene data
Multi gene data can give codon level resolution
What can ribosomal profiling show
Profiling can find ribosomes in new places –> things can be found with ribosomal profiling that would be missed otherwise
Ribosomal profling allows you to ask IF there is no protein are the ribosomes still there
Ribosomal profiling showing ribsomes in new places #1
Found – 95% of genes don’t use the first AUG as a start codon in that ORF (have ORF before it)
- Have an intital ORF in 5’UTR –> ribsoomes see THAT ORF
- Know this because when do ribosomal profiling you can see reads from the intial ORF (Have ribosomes at the main ORF BUT ALSO have ribosomes on the upstream ORF )
- Ribosomes = making a 5 AA long protein that is not used by the cell (could have regulatory effects)
Ribosomal profiling allows you to see that intiatl ORF that is not see by other methods
Ribosomal profiling showing ribsomes in new places #2
Before there was a debate over whether non-coding RNAs are translated
Can use ribosomal profiling to see if they are translated –> shows that if they have a 5’ CAP and polyA tail they will be translated from the first AUG found
Non-Sense mediated decay
IF there is an early stop codon in mRNA = not stable –> it is often recognzid by non-sense mediated decay machinery
These mRNA with premature stop codons might get translated by not as efficientley and the mRNA would not be stable
Ribosomal profiling showing ribsomes in new places #3
They found protein extension in the N terminal
- Shows ORF begins further upstream
Example of Ribosomal profiling show things in the 3’ UTR
For experiment - Have 2 genes in yeast (for BOTH WT or yeast mutant ; WT in black and Mutant in Red)
WT cells = reads the blue ORF and goes to the stop codon (NO black peaks in the 3’ UTR)
Mutant strains – show reads in he 3’ UTR (have red peaks in 3’ UTR)
Overall - Show ribosomal profiling can reveal information about translation
Kinetics of transcriptes
Everything in biology is about rates –> need to look at kinetics of transcripts or the rate of decay
To study = do run on experiment (start to look at a certain time and then follow the process)
- Follow rate (k) in tranlsation
Following K in translation (Run on Experimnet)
Follow rate (k) in tranlsation - want to know if the ribosomes are moving (look at rate of elongation)
Overall - Add heringtonin and wait different times
Process:
1. Start by adding an intiation inhibitor (binds intiating ribosomes to start codons) - Done by adding Harringtonine
- Ribosomes assemble on start site BUT won’t move foward
2. Put drug in cell
3. Allow ribsoomes to run
5. Lyse cells -
6. Trap the ribsomes with elongation inhibitor –> trap what was elongating and get readouts where evyerthing is
7. Look at the rate that the ribosomes run off the mRNA
Can look per cell or gene specific basis (Look at any mRNA and rate of elongation or look locally)
Results of Run on Experimnet
No drugs = black line –> have peak at start and ribsomes across ORF
When add Herringtonine - see ribosomes moving alonng the mRNA:
At 90 seconds the robsomes start moving away from start site on the mRNA
At 120 seconds the ribsomes move foward more
At 150 seconds the ribsomes have tranlsated half the codons
Chart on left - shows time vs. Position (codon) –> shows that the average rate of elongation is 5.6 codons per second (Means E.coli tranlate 20 AA per second)
Second thing Harringtonine run on experiment is used for
Use harrintonin to identify start sites in ALL genes - Have ribosomes marking ALL initiation codons in cell (stuck at initiaon codons in cell)
- See initiation by presence of ribosomes caught by haringtonin)
Top chart - NO Drug = have ribosomal profiling across ORF
Bottom chart - With harringtonine = capture known start site at 5’ end
- Harringtonine shows the major intiation site (upstream of ORF)
- Data shows that there is an upstream ORF and extnsion (Upstream ORF is the primary start site because that is where harringtonin is trapping the intiating ribsomes)
Upstream ORFs
Upstream ORFs = important in biology
Find upstream ORFs using harrington
Initiating Codons
Find intiating codons using Harringtonine
Harringtonine = shows codon composition on mRNA in open reading frame is inition like codons
See normal AUG (most intiations are AUG BUT also see other codons) - have ribosomes that intiatio on non-AUG codons = maybe have other intiation codons
Issue in polysomal and ribosomal profiling
Issue = don’t normalize the the amount of mRNA (just looking to see if there are ribosomes on mRNA) BUT for translational control you want to know if the number of ribosome on the mRNA changes
Might want to know if there are conditions where a given MRNA is more heaviliy translated tan in different conditions
Translational vs. Non-translation control
Non- Translation contol = can have fewer mRNA = feww ribsomes = few proteins
Translation control = same numver of mRNA but fewer ribsomes on them = fewer proteins
- Could have the same number of ribosomes on fewer mRNA (I think can still have more proteins if more ffecicnet)
Graph of Translational vs. Non-translation control
Need to know how much mRNA you have and how many ribosomes are on it
X Axis – mRNA
Y Axis – Proteins/ribsome reads
Left - SHOWS when have transcrtional control - when make more mRNA have more ribsomes (more mRNA = more protein)
Right – shows translational control - if you have the same amount of mRNA BUT have more ribosomal reads
- Increase or decrease the use of mRNA NOT chnaging the amoint of mRNA (just increase the amount of ribsomes loaded onto mRNA)
- Asking about translation efficiency
Translation efficiency
aka ribosomal occupancy - how many ribosomes are on a mRNA
Ribosome Occupancy
Asking how efficiently is a given mRNA recognized by the ribosomes (how many ribsomes are loaded onto mRNA)
X –Axis = mRNA abundace
Y Axis – Translation efficiencey (densisty of ribsome reads per mRNA)
Dots – ALL transcripts in genome
- Blue dots = ribsomal reads of proetin coding –> translation efficncey of 1 = well tranlsated
- Green dots = Ribsomal reads of mRNA in 3’ UTR –> Have lower occupancey values = lower tranlsation effecucey (few ribsomes in 3’ UTR)
- Black dots = lncRNA – some ncRNA re not tranlated well (value similar to 3’ UTR) BUT some are well translated (have many ribosomes on them per density)
Single gene analysis (WT vs. Mutant)
See that on the gene there are more ribosomal reads in the ORF in mutant compared to WT (more read peaks) AND more reads in the the 3’UTR in the mutant compared to the WT
- When knock out the factor in mutant = have ribosomes in 3’ UTR
Meta gene analysis of all of the reads at the stop codon
WT = blue
Mutant = red
Chart shows:
Ribosomes have 3 nucleotide periodicity until reaching the stop codon –> THEN in mutatnt strain there is huge peak of ribosomes at the stop
- See transcriptome wide the accumilation of ribsome reads at the stop (trend occurs in multiple genes)
Issue in meta gene analysis
Issue with meta gene analysis = not counting all of the genes equally because counting reads (1 gene might have more reads)
If have 2 genes where 1 of the gene is made in more abundence then it can bias results (because gene has more input)
Looking at single genes can have power BUT often look at multi genes to see trends
Solution - look at each gene indivdiually
Shows data on a gene by gene basis - Asking if the trend is true for ALL genes are are there a few genes that drive the trend
Looking at ribosomal reads in 3’ UTR:ORF ratio (how many ribosomes are there per length in 3’ UTR relative to the ORF)
- X-Axis = WT ;Y – Axis = mutant
- Each dot = gene
Chart shows that the obersvation is true for MOST genes –> Ratio of ribosome in 3’UTR:ORF is high in WT (dots are above the line)
- Almost all gene shave enriched ribosomes in 3’UTR in mutant
ALL 3 graohs show that the factor that is deleted promotes termination or recylzing because when gone do not have recyling = the ribosome build up on the stop codon
- Have a pile up of stop codons (have polysomes stuck on stop codons = ribsomes stop but not leaving = not being recyled)
Likley is recylcing defect NOT termination defect because if had a temrination dfeect there would still be 3 nucleotide periodicity past the stop codon (ribosome would keep going)
