Lecture #10 (Translation) Flashcards

Question

Role of the Eukryotc Initiation Factors

Answer 1

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

Answer 2

eIF2 (GTPase) = carries the intiatior tRNA - There is a lot of regulation using eIF2

Answer 3

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

Answer 4

1. Selection of correct tRNA (Decoding) 2. Peptide bond formation – AA are ligated to one anotehr 3. Translocation– whole complex is moved along mRNA template to open A site for next tRNA

Answer 5

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)

Answer 6

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

Answer 7

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

Answer 8

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

Answer 9

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

Answer 10

1. P site (peptidyle site) - Has the growing peptide chain 2. A Site (Aminoacyl site) - where trNA are loaded by EFTU 3. E site (Exit site) - where tRNA sits on the way out

Answer 11

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

Answer 12

After termination - Ribosomes Complex is broken apart to allow for next round of translation - Bacteria and Eukaryotes use different factors - Uses ATP

Answer 13

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

Answer 14

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)

Answer 15

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

Answer 16

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

Answer 17

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)

Answer 18

30S, 50S, 70S = bacteria 40S , 60S, 80S = Eukaryotes

Answer 19

Chart is polysome profiles of cells in normal conditions or hypoxic conditions - Shows hypoxic cells = little translation (have 80S BUT no polysomes)

Answer 20

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

Answer 21

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)

Answer 22

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)

Answer 23

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

Answer 24

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

Answer 25

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)

Answer 26

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

Answer 27

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

Answer 28

Peaks = indicates that the ribsomes are slower at that location (have more ribosomes stuck there) ; lower peaks = ribosomes go faster over that location

Answer 29

MOST conclusion = not looking at 1 specific mRNA (not 1 gene) INSTEAD use a meta gene analysis

Answer 30

Chart - shows read size (distribution of the footprint size) --> shows that the ribosome protected regions are 28 nucletodes long

Answer 31

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

Answer 32

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

Answer 33

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

Answer 34

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)

Answer 35

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

Answer 36

Ribosomes translate the right amount of proteins to make protein complexes in the right stochemtry

Answer 37

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

Answer 38

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

Answer 39

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

Answer 40

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

Answer 41

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

Answer 42

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

Answer 43

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

Answer 44

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

Answer 45

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

Answer 46

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)

Answer 47

Power comes from multi gene data Multi gene data can give codon level resolution

Answer 48

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

Answer 49

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

Answer 50

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

Answer 51

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

Answer 52

They found protein extension in the N terminal - Shows ORF begins further upstream

Answer 53

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

Answer 54

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

Answer 55

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)

Answer 56

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)

Answer 57

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)

Answer 58

Upstream ORFs = important in biology Find upstream ORFs using harrington

Answer 59

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

Answer 60

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

Answer 61

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)

Answer 62

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

Answer 63

aka ribosomal occupancy - how many ribosomes are on a mRNA

Answer 64

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)

Answer 65

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

Answer 66

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)

Answer 67

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

Answer 68

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

Answer 69

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)

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Lecture #10 (Translation) Flashcards

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