Test 4 Part 2 Flashcards
Translation in prokaryotes and eukaryotes are pretty much the same true or false?
True
What catalyze is the process of translation?
The ribosome
What directly links, the genetic code in our RNA to the amino acid sequence?
TRNA
So how do tRNAs and ribosomes work together to facilitate the process of translation?
The TRA‘s are going to bind into the ribosome, and then the ribosome will catalyze the formation of the peptide bonds
What is our genetic code made up of?
Codons - it is a series of three bases in the RNA that encode for a specific amino acid
They are located in the open reading frame or the coding sequence, which is a continuous sequence
How will the ribosome’s translate the mRNA?
It will start translation at the start signal and it will read the RNA in a five prime to three prime direction
It will read one codon at a time, adding one amino acid at a time …
It will read that sequence continuously until it gets to the stop signal for translation
What does it mean that our genetic code is non-overlapping?
It means that each base is in one codon
Ex. G(i) is not being shared by Codon 1 and Codon 2 …. It is just in Codon 1 …. Codon 2 may have another G(ii) as part of its code but not G(i)
The benefit to this is that we can add an amino acid and then we can add any of the 20 amino acids right after it
So,
You can have Met, then Met again, the Asp… etc
Vs overlapping which would limit the next codon to whatever the second, and third base is in the first codon which then limits which amino acid comes next
How many possible codons are there?
4^3=64
Why is the code AUG special?
Because it is the start signal for the ribosome telling it where the open reading frame begins so that I can start translating from that position
So in both prokaryotes and eukaryotes, all proteins will begin with a defining amino acid whose codon is AUG
Which three codons do not encode for any amino acid? What is their purpose?
UAA UAG and UGA are our stop codons
They tell the ribosome this is the end of the open reading frame stop translating now
What is reading the codons and bringing in the amino acid that corresponds to that codon?
TRNA
Example of an RNA molecule that does its job in the cell
Like any RNA molecule it is never used directly to make protein
What are the distinct elements of tRNA in its secondary structure?
- At the three prime end of the RNA molecule this is where your amino acid is going to be attached.
- Next is a stem loop structure that sticks out. It is called the T psi C arm (some modified bases here)
(3.) Some tRNA’s have an extra arm between the TpsiC arm and the anticodon arm
- In the anticodon arm, there are three special bases that make up the anticodon. Those bases are going to base pair directly to the codon in the RNA. That is how it knows it has the right codon and that it is bringing in the right amino acid
- Between the amino acid arm and the anticodon arm is the D arm.
Does the tRNA stay in its secondary structure in the cell?
No, it develops a very distinct tertiary structure
The stemloops of the TpsiC arm and the D arm interact with each other forming this tertiary structure…. It looks like an upside down “L”
All tRNA’s have this very specific tertiary structure - this is important for the TRNA‘s to be able to fit into the ribosome
What happens when you are base pairing the tRNA with the mRNA?
Since two RNA molecules are base pairing to each other. They must base pair in an anti-parallel fashion.
So the tRNA is going from five prime to three prime to the left whereas the mRNA is going from five prime to three prime to the right
We are using the anticodon of the tRNA and we are using the codon of the mRNA
Always remember to number positions going in direction from five prime to three prime
What are the biggest determinants for what amino acid is going onto the tRNA? Why?
The first two positions of the codon of mRNA and the second two positions of the anticodon of tRNA
The strongest interactions happen at these two positions
We are doing normal Watson-Crick-Franklin base pairing at these first two positions (codon)
—— the site in the ribosomes that surrounds these bases is very very tight so it only allows for Watson-Crick-Franklin base pairing
What is significant about the third position of the codon of mRNA and the first position of the anticodon of tRNA?
Wobble Position allows for non-canonical base pairing or Non-Watson Crick Franklin base pairing
It is a very unique position in the ribosome the binding site for that base is much looser, which is going to allow for more flexibility in terms of the base pairing that happens there
This allows us to make only 32 tRNA‘s that can recognize all the possible codons
Because of the wobble position, some tRNAs are able to recognize more than one codon because of the wobble position
What happens with tRNAs that can recognize one codon, two codons, or three codons?
One codon means trna can only recognize watson crick only
Two codons : G can base pair with U ,
so U in anticodon means it can pair with Bp with A or G,
Or
If G is in anticodon means it can bp with C or U
Three codons: I can bp with AUC , so if anticodon has I then Codon has A U or C
I is possible because they have a post-transcriptionally added base
What is the second genetic code?
Our first genetic code is the information in the RNA that creates our protein
Our second genetic code are the sequences of the TRNA’s that allows us to ensure the correct amino acid is added onto it
What reads the second genetic code sequence of TRNA’s?
Aminoacyl trna synthetase - reads the second genetic code sequences in tRNA and adds the amino acid onto the TRNA
They will add amino acid onto 3’ end of tRNA
What is the charge versus uncharged TRNA?
If we add an amino acid to a tRNA it is charged
If tRNA doesnt have an amino acid it is uncharged
How do aminoacyl synthetases know they have the correct tRNA?
There are some sequences special to only tRNA’s that are the same for all of them…. It’s what allows them to have the very specific tertiary structure
The sequences that are different are the ones aminoacyl synthetase looks for to make sure it has the right tRNA
The aminoacyl synthetases therefore look for variant bases in the amino acid arm and in the anticodon region of the anticodon arm
How many aminoacyl trna synthetases do we have?
20 …. One for every amino acid
ATP is required for aminoacyl trna synthetase to get the amino acid onto the tRNA
Ex. Of aminoacyl transferase steps to place aa on trna
- It will bind an aa molecule and ATP
- It will transfer the aa molecule onto an AMP and release pyrophosphate in the process (it is a very good leaving group)
- Now it can bind the correct tRNA for that particular aa by looking for the variant bases in the aa arm and anticodon arm
- It will transfer aa onto 3’ end of tRNA
How much of ribosomes are made of RNA and how much is made up of protein?
65% of ribosomes are made of rRNA and 35% protein
RNA is the enzyme for the reaction… protein is for structural purposes
Large subunit is 50s
Small subunit is 30s
Whole ribosome is 70s
Which sub unit does the initiation phase only involve?
The small ribosomal subunit
When does the large ribosomal subunit come in to play?
During the elongation phase
Which proteins prevent the large ribosomal subunit/ from binding before we are ready?
Initiation factors
- iF1 comes in and binds to the 1st site (A) of the ribosome —- it blocks charged trna’s from entering until we’re ready for elongation
- iF3 comes in and binds to the E site which prevents the large ribosomal subunit from binding till elongation
- The ribosome will bind to the 5’ end of that RNA by recognizing its Shine-Delgarno sequence
What is the A site?
Aminoacyl site - all of the charged tRNAs with their aas are going to enter the ribosome through the A site
Where is the Shine-Delgarno sequence in reference to the start codon AUG?
Upstream- it is in the 5’ UTR (this is the sequence we talked about that indicates where ribosome starts) 💖
What does Shine-Delgarno sequence base pair with?
It directly base pairs with the rRNA in the small ribosomal subunit
- base pairing forms hydrogen bonds
Why does the small subunit base pair with the shine delgarno sequence?
Because it positions the start codon AUG in the P site of the ribosome where we need it to be in order to start the process of translation
Is N-formyl methionine in Eukaryotes?
No, F-Met is just in prokaryotes
Very first aa in prokaryotic mRNA sequence
Anywhere else in the sequence is just Methionine
Special tRNA brings in the N-Formyl Methionine
When do we bring in the first tRNA?
We bring in Fmet trna when AUG is in the start site “P”
Fmet TRNA will enter through the P site
IF2 will bind GTP that will give it high affinity for tRNA
IF2 then binds to tRNA and brings it into the the P site where its anticodon will base pair with the start codon
—- once base pairing happens then IF2 knows translation is ready to begin
IF2 is going to hydrolyze its GTP which will subsequently cause all the initiation factors to fall off
IF3 is gone so the large ribosomal (50s) subunit will bind
—— we have now ended initiation and formed our transition initiation complex
Please look at future card for elongation process
Which tRNAs enter through the Psite?
Only Fmet tRNA
Where do the uncharged trna’s go during translation?
To the E site “exit” site
What are elongation factors?
Proteins involved in elongation or EFs
EF-Tu first binds to GTP then brings the charged tRNAs to the A site
The anticodon of that tRNA will base pair with whatever the second codon is …. Once the base pairing has happened then EF-Tu will hydrolyze GTP and leave
Now the FMet TrNA is in the P site and the second TrNA is in the A site so, now we are ready to make a peptide bond
Fmet and the aa on 2nd tRNA will form a peptide bond which is catalyzed by the rRNA molecule peptidyl transferase which causes the amino end of the aa in the A site to attack the aa in the P site which forms the peptide bond between them……
——- but you end up breaking the bond of the aa in the P site with its tRNA at the 3’ end of its tRNA
So we move onto translocation… look on future card …
True or false we recycle EF-Tu
True.
EF-Ts binds to things and tells them to let go of one nucleotide and bind a different nucleotide. So, it is instrumental in exchanging nucleotides
So, it binds EF-Tu that has GDP bound to it and it tells EF-TU to let go of GDP…. This allows EF-TU to bind a different nucleotide, GTP so it can bind another trna and repeat its process
What is involved in Ribosome Translocation?
Elongation factor EFG will bind GTP near the A site
It will use the power of GTP hydrolysis to kick all the trNa’s one site over
The only thing in the A site is the 3rd codon
The Peptidyl “P” site now has a dipeptide in it that is on the tRNA there….. this is where the growing polypeptide chain is going to be
Then the uncharged trna will go to the e site and leave
This process will repeat for every single codon till we get to a stop codon which stops translation
Look for future card on translation termination
What happens during Translation Termination?
When ribosome gets to a stop codon it sits and waits till a tRNA comes in
Release Factors or Termination Factors will recognize the stalled ribosome and bind into its A site
The Termination Factors will then trick the ribosome because the ribosome senses something in its A site so the ribosome tries catalyzing a peptide bond but ends up reacting with water which promotes a hydrolysis reaction
So, it hydrolyzes the bond between the 3’ end of the tRNA in the P site and the peptide chain which ends translation
What do ribosome recycling factors do? (RRFs)
Bind to the ribosomal complex and force it to dissociate …. So we can reuse all these parts together and start translation again
