Lecture 19 - Translation (pt 2) Flashcards
3 sites on ribosome and the corresponding tRNA
A site (Amino acyl) for aminoacyl tRNA. P site (Peptidyl) for Peptidyl tRNA E site (Exit)
How peptide bond between 2 a.a forms in ribosome and where goes the polypeptide chain
N on N-terminal part of the next a.a. attacks the carbon on the carboxy terminal part of the previous a.a. Chain goes on the amino acyl tRNA. It moves to the P site and moves the mRNA with it and becomes the peptidyl tRNA
Structure of the ribosome and important thing to remember about process of translation discussed
One large subunit and one small subunit. Translation discussed is about protein synthesis in EUKARYOTES.
Why translation is complex
Because you have to get the specific sequence for each protein
What initiation phase of eukaryotic translation consists of
Assembly of ribosome with an mRNA and an initiator tRNA charged with methionine
What elongation phase of eukaryotic translation consists of
Stepwise addition of a.a. to the polypeptide chain
What termination phase of eukaryotic translation consists of
Release of completed polypeptide and release of ribosome. Disassembly of the ribosome.
What tRNA is needed to start synthesis of the polypeptide chain
tRNAi Met
Two types of tRNAs for the methionine codon, their function and their specific aminoacyl tRNA synthetase
1) tRNAi Met (initiator)
2) tRNA Met (regular one - for Met in middle of the protein)
Charged with the same aminoacyl tRNA synthetase
tRNA i Met in eukaryotes, archaeans and bacteria
Eukaryotes and Archeans -> tRNA i Met + methionine —> Met - tRNA i Met
Bacteria -> tRNA i Met + methionine + FORMYL GROUP (CHO) —-> fMet-tRNA i Met
Something particular about the chemical modifications of the first methionine in bacteria
It can be recognized by our cells as foreign.
tRNA Met (regular one) in eukaryotes, archaeans and bacteria
tRNA Met + methionine —> Met - tRNA Met
What happens to Met when protein synthesis is finished
All proteins start with Met when they are made but Met can be changed/removed later.
Formation of 43 S preinitiation complex - 1st step
tRNA i - Met (charged with Met ) binds eIF2 carrying GTP to form the eIF2 ternary complex
T/F : mRNA joins 43 S preinitiation complex
False . No mRNA at this step
What eIF# stands for
eukaryotic initiation factor 1,2,3, etc.
What is a GTPase (2 things)
- small molecules that can have 2 shapes depending on if they bind GTP or GDP
- Can hydrolize GTP
eIF2 precise function. Does it take part in the complex ?
Not part of the translation machine but acts as a regulatory component
Important concept in translation
Regulatory checkpoints
What 40 S contains (4)
- Small ribosomal subunits with E, P, A site
- eIF3 that binds to small ribosomal subunit near E site
- eIF1 binds E site
- eIF1A binds A site
Formation of 43 S preinitiation complex - 2nd step
eIF2 ternary complex, 40 S and eIF5 join to form 43 S complex
What 43 S complex contains
40 S with 1) ternary complex (tRNAi - Met bound to eIF2 - GTP) on the P site
and 2) eIF5 bound to eIF1 (that is bound to E site)
How can protein synthesis be negatively regulated and how it works
phosphorylation of eIF2 on different residues makes it keep GDP. GDP and GTP bind the same site on it so it affects its ability to bind GTP (switch from GDP to GTP = becomes active)
What eIF4 complex contains
4E, 4G, 4A (and 4B - when it binds the mRNA)
What mature mRNA ready for translation initially contains
3
1) 5’ cap
2) Start and stop codon
3) Poly(A) tail bound by PABPC
How mRNA activation works
eIF4 complex (and 4B) binds to mRNA
Near what region eIF4 complex binds on mRNA
near 5’ cap
What eIF4E binds to
5’ cap structure
What eIF4G binds to
PABPC (which is bound to Poly(A) tail)
What eIF4A and eIF4B do
eIF4A RNA helicase unwinds RNA secondary structure at 5’ end. eIF4B joins and stimulates helicase activity
Note about secondary structure at 5’ end of mRNA
Not necessarly present
Why does mRNA activation reaction (eIF4 complex (and 4B) binds to mRNA ) requires ATP/phosphorylates ATP
ATP is required to unwind RNA secondary structure at 5’ end if present, by eIF4A RNA helicase
Attachement of 43S preinitiation complex to mRNA how it happens
eIF3 (bound to small ribosomal subunit near E site) interacts with eIF4 (on 5’ region of mRNA)
Step after Attachement of 43 S preinitiation complex to mRNA and how it works
Scanning from 5’ to 3’. mRNA is pulled in the groove of the small ribosomal subunit until tRNAi sees AUG.
What happens after recognition of AUG by tRNAi and what does that do
eIF2 recognizes that this happened and hydrolizes GTP to GDP (release of Pi). This causes scanning to stop.
What is 48 S intitiation complex.
43 S with the mRNA, the tRNA i recognizing the AUG and eIF2 bound to GDP
Recruitment of large ribosomal subunit. Its name, its regions and what happens when it binds 48 S
60 S. Regions complementary to E, P and A site. When it binds 48 S, 5B-GTP binds 1A (bound to A site) and displacement of eIF1, 2-GDP, 3, 4 (E,G,A and B) and 5
What is left after recruitment of large ribosomal subunit (4)
2 ribosomal subunits with E, P and A site.
RNAi - Met recognizing AUG at P site.
1A bound to A site and 5B-GTP.
The mRNa.
What steps follows large ribosomal subunit binding (2). What’s the name of the complex formed. What this causes
- Hydrolysis of GTP on eIF5B.
- Release of eIF5B bound to GDP and release of eIF1A
This forms the 80 S initiation complex. It can no longer dissociate and is committed to translation
Condition for hydrolysis of GTP on eIF5B
Large ribosomal subunit must bind correctly (fits right). Other large subunit that don’t fit right come and leave. Hydrolysis of GTP on eIF5B marks the correct fitting and dissociation can no longer happen.
What the 80 S initiation complex contains
- Ribosome and its 3 sites E, P, A
- mRNA
- tRNA i Met - Met recognizing AUG
After 80 S initiation complex is obtained, what triggers translation elongation
Entry of charged tRNAs bound to EF1alpha ( ELONGATION FACTOR 1 ALPHA) on A site. When right tRNA enters, GTP on EF1alpha is hydrolized
GTP on EF1alpha hydrolysis reaction and what happens to the ribosome (2)
EF1alpha-GTP –> EF1alpha-GDP + Pi.
These products are RELEASED and this causes
1) a conformational change in the ribosome and
2) a reposition of the tRNA on A site -> tRNAs become adjacent and peptide formation can start
What catalyzes the first peptide bond formation. What is the reaction called and what is the product obtained and what does it contain.
Catalyzed by large ribosomal RNA. Reaction called peptidyltransferase reaction. Oligopeptide obtained contains Meti and aa2
After first peptide bond formation, translocation happens. (and happens after each peptide bond formation). What happens during translocation
Ribosome moves along mRNA a distance of one codon.
tRNA i moves to E site, tRNA aa2 moves to P site, A site is vacant.
What is necessary for translocation to happen (it is essential a physical displacement in the ribosome)
EF2 - GTP must recognize successful elongation. It hydrolyzes GTP and EF2 - GTP + Pi is obtained. This makes the step irreversible
First step of translation termination
No tRNA binds the stop codon. Instead, eRF1 (eukaryotic release factor 1) bound to eRF3-GTPase goes to the A site.
Second step of translation termination
eRF3 - GTP HYDROLYSIS acts with eRF1 to promote cleavage of the peptidyl-tRNA, releasing the finished polypeptide. (Pi is obtained)
What do we call the complex formed after release of the polypeptide and what is bound to eRF1 at this point
Post-termination complex. eRF3-GDP is bound to eRF1.
How are the subunits of the ribosome and the mRNA all seperated
IN A PROCESS USING ATP (ATP - ADP + Pi), ABCE1 binds the post-termination complex and separates the ribosomal subunits
What happens to small ribosomal subunit after seperation of the ribosome (what is released (3) and what binds to it (3))
P tRNA, eRF1 and eRF3-GDP are released (they’re all seperated)
eIF1, eIF1A and eIF3 bind to small ribosomal subunit again.
Elongation rate characteristics (2)
- Relatively constant elongation rate.
2. Typical protein molecule takes 30-60 sec. to synthesize
What do we call multiple ribosomes
polysomes
First way of maximizing rate of translation
Simultaneous translation from the same mRNA by multiple ribosomes (polysomes)
Second way of maximizing rate of translation
Efficient reinitiation and efficient recycling of ribosomal subunits
How can the mRNA take a circular form during translation
Interaction of factors that bind the 5’ (eIF4G) with factors that bind the 3’ Poly(A) tail (PABPC)
How circular shape of mRNA can make reinitiation and ribosomal subunits recycling more efficient
Upon termination, ribosome subunits will be close to the mRNA 5’ end (this enhances reinitiation)
Names of the large and small ribosomal subunits when they are released and before any translation initiation
Small ribosomal subunit = 40S
Big ribosomal subunit = 60S
What allows monitoring and checkpoints of individual steps in translation
GTPases that hydrolize GTP and cause irreversible conformational changes in the complex.
