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MB Lec 15: The Mechanism of Translation 1 (Initiation)
Terms in this set (55)
Definition of initiation
The rate limiting step (the step that takes the longest time in translation).
Similarities b/w eukaryotic and prokaryotic translation initiation
1.) Both start with a mRNA
2.) Both involves ribosomes, however, some of the protein factors are different.
Before prokaryotic initiation of translation can begin:
1.) tRNA must be "charged" to provide aminoacyl-tRNAs
2.) Ribosomes must be dissociated into subunits because initiation complex forms on the small ribosomal subunit.
All tRNAs have what kind of conserved sequence at their 3' end?
CCA conserved sequence at 3'end.
The CCA sequence is added AFTER the tRNA is synthesized.
Definition of ester bond
A covalent bond between an amino acid and an alcohol (OH group) with the release of H20.
In a tRNA, the ester bond that is between the CCA 3'OH and its corresponding amino acid is catalyzed by which enzyme?
aminoacyl-tRNA synthetase catalyzes the formation of that ester bond.
Note: There are 20 versions of this enzyme as we have 20 different amino acids, each specific synthetase recognizes a specific type of amino acid.
What type of bond is required to attach an amino acid to a t-RNA?
ester bond (which is actually a type of covalent bond)
What determines the specificity of a tRNA to an amino acid?
The enzyme that links a specific amino acid to the tRNA CCA 3'OH end is a specific type of aminoacyl-tRNA synthetase.
Describe the 2 steps in tRNA charging
Step 1: Aminoacyl-tRNA synthetase couples an amino acid to AMP (from ATP) to form aminoacyl-AMP. Byproduct = pyrophosphate
Step 2: Synthetase replaces the AMP with tRNA to form aminoacyl-tRNA. Byproduct = AMP
What are the prokaryotic ribosomal subunits?
30S + 50S = 70S
What are the eukaryotic ribosomal subunits?
40S + 60S = 80S
Which ribosomal subunit initiates translation?
It's the small ribosomal subunit (30S in the case of eukaryotes).
What are the 3 initiation factors (IFs) of E.coli (prokaryotes)?
IF-1, IF-2, and IF-3
Each one of these bind to the ribosome. IF-3 binds more strongly to the ribosome.
How were the 3 prokaryotic initiation factors (IF-1, 2 & 3) identified?
They were identified by washing ribosomes with high salt buffer. IF-1 and IF-2 were removed with 0.5M of NaCl and IF-3 was removed with 1M of NaCl. Again, this shows that IF-3 binds more strongly to the ribosome.
What is EF-G and what is it's function?
EG-G = Elongation Factor G
Function = involved in elongation. Both EF-G and RRF promote the dissociation of ribosome subunits.
What is RRF and what is it's function?
RRF = Ribosome Release Factor
Function = involved in the release of the ribosome from a transcript (mRNA) at the end of translation. Both EF-G and RRF promote the dissociation of ribosome subunits.
Prokaryotic Ribosome Composition
LARGE: 50S = 23S + 5S + 31 proteins
SMALL: 30S = 16S + 21 proteins
50S + 30S = 70S
Eukaryotic Ribosome Composition
LARGE: 60S = 28S + 5.8S + 5S + 50 proteins
SMALL: 40S = 18S + 33-35 proteins
60S + 40S = 80S
How does IF-3 prevent the 50S from reassociated with the ribosome?
Once ribosome subunits dissociate with the help of RRF & EF-G, IF-3 binds to the free 30S in order to block the 50S from reassociating with the 30S.
All 3 prokaryotic initiation factors bind near which end of the 16S rRNA?
All 3 IFs bind to the 3'end of the 16SrRNA (which is part of the 30S subunit.
All 3 prokaryotic IFs bind to which rRNA subunit?
All 3 IFs bind to the 16S rRNA which is part of the 30S subunit.
When all 3 IFs bind to the 16S rRNA (part of 30S subunit), this binding attracts the binding of what?
Binding of all 3 IFs to the 16S rRNA attracts binding of mRNA and charged aminoacyl-tRNA.
The first methionine amino acid that gets attracted and bound to the 30S (once all initiation factors bind to the 3' end of the 30S) is modified in what way?
The first methionine amino acid that binds to the 30S is actually formylated. Thus, it's a N-formyl-methionine.
How was N-formyl-methionine discovered?
1.) Take charged t-RNA and digest it with a nuclease (ex: RNase).
RESULT: You get a bunch of nucleotides plus the charged aminoacyl-tRNA.
Conclusion: This shows the digested products of any typical charged aminoacyl-tRNA.
2.) Take a tRNA-CCA-methtionine and digest it with RNase to see it's composition.
RESULT: You get a bunch of chewed up nucleotides + adenyosyl-methionine + adenosyl-N-formyl-methionine.
Conclusion: This shows the digested products of a charged Met-tRNA and thus, fMET was discovered in the byproducts of this reaction.
The formyl group in fMET is added to what part of the methionine amino acid?
The formyl group is added to the amide of your Met amino acid.
Which enzyme adds the formyl group to the amide of Met?
When does formylation of Met take place?
Formylation of Met only happens when tRNA is already charged with methionine.
What's the purpose of fMet-tRNAs being placed at the start of a peptide?
It serves as an initiating aminoacyl-tRNA.
When is N-formyl-methionine removed from the protein?
It's removed from a mature protein, thus it gets cleaved off AFTER translation.
What are the 3 initiating codons and what amino acids do they code for?
AUG = codes for Met
GUG = codes for valine
UUG = codes for leucine
TRUE or FALSE?
Shine-Delgaro Sequence is only found in prokaryotes?
What is the Shine-Delgarno Sequence?
Where is the Shine-Delgarno Sequence located?
It's located just upstream of the start codon where all or part of its AGGAGGU sequence resides.
The Shine-Delgarno Sequence is complementary to what sequence?
It's conserved AGGAGGU sequence is complementary to the sequence at the 3'end of the 16S rRNA.
What is the importance of the 16S rRNA basepairing with the Shine-Delgarno sequence?
The ability of the 16S rRNA to basepair with the Shine-Delgarno sequence upstream of the initiation start site is essential for:
1.) ribosome binding and
2.) translation of mRNA
TRUE or FALSE?
Prokaryotic mRNAs are polycistronic
TRUE; They can have multiple ORFs that initiate independently.
Thus, you can have multiple independent Shine-Delgarno sequences on the mRNA, where each cistron would have its own ribosome binding site and initiation codon.
A section of a DNA or RNA molecule that codes for a specific polypeptide in protein synthesis.
Define compensatory mutation
Mutations that correct a loss of fitness due to earlier mutations.
What factors are involved in binding mRNA to the 30S subunit?
Binding is primarily mediated by IF-3 and further increased by IF-1 and IF-2.
NOTE: IF-2 is responsible for binding fMet-tRNA to the 30S subunit, but all 3 initiation factors together yield optimum binding.
TRUE OR FALSE?
1 mole of GTP binds to 30S for every 1 mole of fMET-tRNA that gets bound to the 30S.
TRUE OR FALSE?
1 mole of GTP is required for IF-2 binding at physiological concentrations.
TRUE; This means that if you artificially increase [IF-2] then it will bind to the 30S without GTP.
NOTE: GTP does not get hydrolyzed when IF-2 binds. It gets hydrolyzed when IF-2 leaves (so when 50S joins 30S).
How is GTP removed when 50S joins the complex?
IF-2 has its own GTPase activity (hydrolyzes GTP to GDP +Pi). IF-2 will only hydrolyze GTP when 50S joins 30S. This activity is ribosome-dependent.
What is the function of GTP hydrolysis?
To allow removal of IF-2 from the ribosome (and also GTP itself) so that the 70S complex can begin elongation of the peptide chain.
Describe the steps in how translation initiation occurs in prokaryotes.
1. Dissociation of 70S into 50S and 30S with the help of RRF and EF-G.
2. Binding of IF-3 to 30S to prevent re-association with 50S.
3. Binding of IF-1, IF-2 with GTP alongside IF-3.
4. Binding of mRNA and fMet-tRNA to form 30S initiation complex. IF-2 is mostly responsible for fMet-tRNA and IF-3 is responsible for mRNA binding.
5. Binding of 50S, loss of IF-1 and IF-3.
6. Loss of IF-2 with hydrolysis of GTP. 70S initiation complex can begin elongation.
NOTE: look at diagram on slide 15 of lec 15.
What are the differences between eukaryotic and prokaryotic translation initiation?
2 main differences:
1.) Eukaryotes usually start translation peptide with Met whereas prokaryotes start it with fMet.
2.) Eukaryotes have no SD sequence in which prokaryotes have. Rather, eukaryotes use a scanning mechanism for ribosome initiation. This mechanism occurs when initiation factors (proteins) bind to CAP to help guide the ribosome to the 5'end.
What is the scanning mechanism in eukaryotes?
40S small eukaryotic ribosome subunit binds to CAP with the help of initiation factors that attract it to the 5'end. Then the 40S will scan the sequence until it finds an AUG, which at that point it starts translation.
Who discovered the scanning mechanism of eukaryotes?
Marilyn Kozak (a Biochem prof who also discovered the Kozak Consensus Sequence).
What are the 4 considerations in the scanning model?
Marilyn Kozak's scanning model is based on 4 considerations as followed:
1.) In no instance was translation initiated on an internal AUG. => Ribosomes don't initiate translation at an internal AUG.
2.) Initiation didn't occur at a fixed distance from the 5' end. => Sometimes mRNAs can have very long 5'UTR or very short 5'UTR. So there's variability among mRNAs in eukaryotes. Thus, unlike in prokaryotes with their fixed distant SD sequence, eukaryotes can have their first AUG at any distance from the 5'end. Thus, ribosomes don't measure a certain distance from the 5'end in order to initiate translation.
3.)The first AUG downstream of the cap was used for initiation. => However, in about 5-10% of all mRNAs, the ribosome doesn't initiate at the first AUG. It needs to find the correct AUG.
4.)The CAP at the 5'end facilitates translation.
What is the ideal AUG for the eukaryotic scanning mechanism?
The ideal initiating AUG is one that is surrounding by a Kozak consensus sequence.
What is the Kozak Consensus Sequence, and which variety of it is the best kind?
R = purine (A or G, however, the best would be if R = A)
What is the translation initiating sequence for prokayotes?
AGGAGGU = SD sequence
What is the translation initiating sequence for eukaryotes?
CC(R)CC[AUG](G) = Kozak consensus sequence
TRUE OR FALSE?
If you have the most ideal kozak consensus sequence, then moving it upstream and out of frame should force the ribosome to begin translation out of frame?
What are the effects of a secondary RNA structure on initiation?
The shape of 5' UTR can have a secondary RNA stemloop structure.
Sometimes this can have a positive effect because if the stemloop is 15nt downstream of the AUG, then it tends to pause the ribosome to make it re-think and determine if the AUG is in a weak surrounding sequence or not.
The stemloop at the 5' end could also have a negative effect where it is placed too close to the CAP and so the ribosome doesn't have enough room to bind to the CAP. Or the stemloop is too large which blocks the ribosome from going through it.
What are the 4 different secondary RNA structures (stemloops) that can be present at the 5'UTR. Describe the effect each structure has on initiation.
These are the 4 different secondary constructs that Kozak created.
Construct #1 shows that putting the stemloop too close to the 5'UTR will not allow the ribosome to bind to CAP, thus, you get no translation.
Construct #2 shows that if AUG is within the stemloop but is far from CAP (thus, giving the ribosome enough space to bind) then you can still get some translation. NOTE: ribosome has helicase activity so it will unwind the secondary nature of the stemloop in order to access the AUG and scan through it.
Construct #3 is the best one as it allows for the most translation (has no stemloop, thus no barrier).
Construct #4 shows that having too large of a stemloop will block the ribosome from scanning to the AUG, hence no translation.
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