II. Post-transcription | 26. Elongation and termination of pro- and eukaryotic translation; pharmacological inhibitors of translation Flashcards
- Basics
a/ What are the 3 stages of translation?
(1) initiation, (2) elongation, (3) terminatio
- Basics
b/ What is the first step of the initiation stage in both pro- and eukaryotes?
the first step of the initiation stage is the binding of the tRNAiMet and the mRNA to the small ribosomal subunit.
- Basics
c/ Proteins are synthesized in which direction?
N -> C direction
( The N-terminal is the amino acid reside on one end with an amine group, while the C-terminal is the amino acid residue on the other end with a carboxylic group.)
- Eukaryotic elongation
a/ What is the role of translation elongation factors (EFs)?
A set of special proteins, called translation elongation factors (EFs), are required to carry out the process of chain elongation.
- Eukaryotic elongation
b/ What are the 2 key steps in elongation?
(1) entry of each succeeding aminoacyl-tRNA with a tRNA complementary to the next codon = formation of peptide bond
(2) the movement (translocation) of the ribosome,
one codon at a time along the mRNA
- Eukaryotic elongation
c/ What are the steps in elongation?
1) At the completion of initiation, tRNAiMet is bound to the P-site on the assembled 80S ribosome. The 2nd aminoacyl-tRNA is brought into the ribosome as a ternary complex (tRNAiMet + eIF2 bound to GTP) in association with EF1α-GTP -> becomes bound to the A-site
2) Translation can only proceed if the right aminoacyl-tRNA- EF1α-GTP complex arrives. If it does, the GTP will be hydrolyzed -> release of EF1α-GDP -> tRNA binds tightly
3) Amino group of the 2nd amino acid reacts with the activated methionine on the initiator tRNA, forming a peptide bond catalyzed by the large subunit
4) Hydrolysis of GTP in EF2-GTP causes another conformational change in the ribosome that results in its translocation of one codon along the mRNA and shifts the tRNAiMet to the E-site and the tRNA (with the bound peptide) to the P-site
5) The cycle can begin again with binding of a ternary complex bearing the 3rd amino acid to the now open A-site
-> Since both EF1α and EF2 have GTP-activity, each amino
acid that attaches to the growing peptide requires 2 GTP
- Prokaryotic elongation
a/ What are the steps in prokaryotic elongation?
1) Elongation starts when fMet-tRNA enters the P- site, causing conformational change which opens the A-site for the new aminoacyl-tRNA to bind. Facilitated by EF-Tu (same as EF1α)
2) Peptide bond formation is catalyzed by the large subunit of prokaryote (50S)
3) Translocation is catalyzed by EF-G (same as EF2), which shifts the fMet-tRNA to the E-site and the tRNA (with the bound peptide) to the P-site
4) The fMet-tRNA is released from the ribosome, and a new tRNA can occupy the A-site
-> Ribosome continues to translate remaining
codons on the mRNA, until it reaches a stop codon on the mRNA (UAA, UGA or UAG)
- Difference between prokaryotic and eukaryotic elongation
Basically the same process as in the eukaryotes, but with different EFs (NOTE: all EFs here also have bound GTP as in the eukaryotes = same reactions. Just not written again)
- Eukaryotic termination
a/ What does termination require?
Termination requires highly specific molecular signals that decide the fate of the mRNA- ribosome-peptidyl-tRNA complex.
- Eukaryotic termination
b/ What are the 2 types of specific protein release factors (RFs) for eukaryotic termination?
- eRF1: shape is similar to that of tRNA, acts by binding to the ribosomal A-site and recognizes the stop codon correctly
- eRF3: is a GTP-binding protein. The eRF3-GTP complex acts together with eRF1 to promote cleavage of the polypeptide chain from peptidyl-tRNA
- Eukaryotic termination
c/ What are the steps in eukaryotic termination?
1) eRF1 recognizes the stop codon correctly
2) peptidyl-tRNA bond of the tRNA in the P-site is cleaved, resulting in termination of translation
3) Release of the completed protein leaves a free tRNA in the P-site and mRNA will still associate with the 80S ribosome (with eRF1 and eRF3- GDP bound in the A-site)
4) Ribosome recycling occurs when this post-termination complex is bound by a protein called ABCE1, which uses energy from ATP hydrolysis to separate the subunits and release the mRNA
-> Initiation factors eIF1, eIF1A and eIF3, which are also required for separation of the subunits, load onto the 40S subunit, making it ready for another round of initiation
- Eukaryotic termination
c/ What are the steps in eukaryotic termination?
1) eRF1 recognizes the stop codon correctly
2) peptidyl-tRNA bond of the tRNA in the P-site is cleaved, resulting in termination of translation
3) Release of the completed protein leaves a free tRNA in the P-site and mRNA will still associate with the 80S ribosome (with eRF1 and eRF3-
GDP bound in the A-site)
4) Ribosome recycling occurs when this post-termination complex is bound by a protein called ABCE1, which uses energy from ATP hydrolysis to separate the subunits and release the mRNA
-> Initiation factors eIF1, eIF1A and eIF3, which are also required for separation of the subunits, load onto the 40S subunit, making it ready for another round of initiation
- Prokaryotic termination
The ribosome continues to translate the remaining codons on the mRNA as more aminoacyl-tRNA bind to the A-site, until it reaches the stop codon on mRNA (UAA, UGA, UAG)
1) The stop codon moves into the A-site
2) Since there are no stop codon-recognizing tRNA in prokaryotes, they will be detected by RF1 or R2 (releasing factors)
3) RFs trigger the hydrolysis of the ester bond in the tRNA-peptide and release the synthesized protein from the ribosome
4) RF3 (GTPase activity) makes both RF1 and RF2 leave the ribosome
5) RFF (ribosome recycling factor) is involved in the ribosome recycling, which will release mRNA and tRNAs from the ribosome + dissociate the 70S into 50S and 30S
- How is peptide bond formed?
In P site there is the growing peptide chain and in the next AA comes via tRNA to the A site. Then the new AA will attack the ester link and forms new peptide bond and then the OH group is released again.
- the function of ribosome is the oriented in a proper way the incoming tRNA with the growing peptide chain in the p site.
- The OH group of the tRNA is the real catalyse in the reaction which can be involve is proton movement from amino group to hydroxyl group.
- All proteins are synthesized in N to C direction.
- Pharmacological inhibitors of translation
a/ What are the 2 translation toxins?
- Diphetria
- Ricin
- Pharmacological inhibitors of translation
b/ Describe Diphteria
- The phage infected Corynebacterium diphteriae produce the toxin
- Few μg could be lethal, a single toxin molecule can kill the cell
- It catalyzes the ADP ribolysation of diphtamide of EF2
- Pharmacological inhibitors of translation
c/ Describe Ricin
- In the seeds of Ricinus communis
- 500 μg could be be lethal
- Due to its N-glycosidase activity, it cleaves a certain adonosine of 28S rRNA
- Pharmacological inhibitors of translation
d/ What are the examples of antibiotics that can Interfere with the small subunit? (of prokaryotes)
- Tetracycline: blocks binding of aminoacyl-tRNA to the A-site
- Streptomycin: prevents transition from initiation to chain elongation + causes miscoding
- Spectinomycin: binds to 30S
- Hygromycin B: stabilizes A-site, which result in inhibiting translocation
- Pharmacological inhibitors of translation
e/ What are the examples of antibiotics that can Interfere with the large subunit? (of prokaryotes)
- Chloramphenicol: prevents formation of peptide bonds by interacting with 50S subunit
- Streptogramin B/A: binds to P-site of the 50S
- Erythromycin: binds in the exit channel of the ribosome, thereby inhibiting elongation of peptide chain
- Pharmacological inhibitors of translation
f/ An example of antibiotic that can inhibit protein synthesis in. both prokaryotes and eukaryotes
Puromycin: Aminoacyl-tRNA mimicry
- Pharmacological inhibitors of translation
g/ What is the role of Chloramphenicol?
- Chloramphenicol: prevents formation of peptide bonds by interacting with 50S subunit
!! examples of antibiotics that can Interfere with the large subunit? (of prokaryotes)
- Pharmacological inhibitors of translation
h/ What is the role of Streptogramin B/A?
Streptogramin B/A: binds to P-site of the 50S
!! examples of antibiotics that can Interfere with the large subunit? (of prokaryotes)
- Pharmacological inhibitors of translation
i/ What is the role of Erythromycin?
Erythromycin: binds in the exit channel of the ribosome, thereby inhibiting elongation of peptide chain
!! examples of antibiotics that can Interfere with the large subunit? (of prokaryotes)
- Pharmacological inhibitors of translation
j/ What is the role of Tetracycline?
- Tetracycline: blocks binding of aminoacyl-tRNA to the A-site
!!! examples of antibiotics that can Interfere with the small subunit? (of prokaryotes)
- Pharmacological inhibitors of translation
e/ What is the role of Streptomycin?
Streptomycin: prevents transition from initiation to chain elongation + causes miscoding
!!! examples of antibiotics that can Interfere with the small subunit? (of prokaryotes)
- Pharmacological inhibitors of translation
e/ What is the role of Spectinomycin?
- Spectinomycin: binds to 30S
!!! examples of antibiotics that can Interfere with the small subunit? (of prokaryotes)
- Pharmacological inhibitors of translation
e/ What is the role of Hygromycin B?
Hygromycin B: stabilizes A-site, which result in inhibiting translocation
!!! examples of antibiotics that can Interfere with the small subunit? (of prokaryotes)
