Lecture 17 - Translation II Flashcards
Describe the basic of translation in prokaryotes and eukaryotes.
Translation in Prokaryotes
In prokaryotes there can be a number of proteins encoded by the same mRNA (polycistronic) and that a number of ribosomes ca work their way down one mRNA, which has just been formed by transcription.
In prokaryotes transcription and translation is coupled - can happen at same time and location.
Translation in Eukaryotes
Methionine is the first amino acid (but isn’t formylated) and there is a special form of initiator tRNA that only binds to the first AUG codon.
No Shine Dalgarno sequence on mRNA - instead the 7-methylG cap assists in binding of ribosome and ribosome moves along to the first AUG
The preferred sequence context for start codons in mammals is RXXAUGG (the Kozak sequence)
Describe Eukaryotic mRNA.
The 7-methylguanosine cap and polyA-tail stabilise mRNA and prevent degradation.
Note: not all the mRNA acts as a template - there are both 5’ and 3’ untranslated regions (UTRs) (found at both 5’ and 3’ end) the 3’ end particularly is important in stability
What are Eukaryotic Initiation Factors.
As with the prokaryotic system specific Initiation Factor proteins (eIFs) are needed to assist at the start of translation - allow ribosome to bind.
The system is more complex with 12 eIFs identified. eIFs are numbered 1-6, but each number is a group of different types and are further named with a letter such as eIF4A and eIF4B etc.
1. The mRNA is bound by the eIF4 family of initiation factors. eIF4E recognises the cap and acts as a scaffold between the cap and the polyA tail forming a closed loop complex - PolyA binding proteins bind to 2' end - critical in interaction with eIF4G. The small subunit is bound by both the initiator tRNA and several initiation factors also recruited via eIF4G/eIF3 interaction. - eIF3 is important as it links the ribosomal complex with 4G linking them and forming the pre-initiation complex. 2. The pre-initiation complex scans for the start codon: the eIF4A/4B complex has ATP-dependent helicase activity - drawing the mRNA through until the start codon is located. The 4a and 4B can then dissociate. 3. Once a suitable start codon has been identified the GTPase activity of eIF2 is activated causing a conformational shift. This then allows the large subunit to bind and the majority of the remaining eIFs are released. 4E and 4G remain keeping the loop structure.
Describe eukaryotic elongation
Elongation is very similar between eukaryotes and prokaryotes
* eEF1A is equivalent to EFTu - binds aminoacyl tRNA and GTP
* eEF1b is equivalent to EFTs and exchanges GTP for GDP
eEF2 is equivalent to EFG and is involved in ribosome translocation
Describe termination in eukaryotes.
Very similar to prokaryotes
However the only release factor eRF1 recognises all termination codons - mimics the structure of tRNA. In prokaryotes there is two release factor. The release factor is already bound to eRF3 which has intrinsic GTPase activity when bound to stop codon GTP changed to GDP and causes dissociation pf eRF3.
Polypeptide chain is still attached at this point
ABCE1 another accessory protein an ATPase binds to eRF1 and causes hydrolysis of bond holding polypeptide chain. ATP activity activated and ADP formed which releases energy needed for release of polypeptide. eFR1 and tRNA can then dissociate
eIF1 and eIF3 region small rRNA subunit.
The ribosome can then be recycled.
How do protein synthesis inhibitors act as antibiotics.
- A range of compounds have been shown to inhibit translation on prokaryotic 70S ribosomes but not on eukaryotic 80S ribosome. Antibiotics can target ribosomes in bacterial cells specifically.
- These compounds prevent the growth of bacteria and should not affect human and animal protein synthesis.
- But 70S ribosomes are also present in mitochondria, thus some antibiotics can have detrimental effects.
Kasugamycin -
Tetracycline - stops charged tRNAs from entering A site
Kirromycin - Prevents the release of eFTu
Aminoglycosides - cause the ribosome to be less accurate so the wrong tRNA introduced
Chloramphenicol - prevents bond forming between amino acids
Thiostrepton - Stops translocation
Erythromycin - Binds to exit of ribosome so blocks protein release.
What is Fusidic acid
Fusidic acid is a narrow spectrum steroid antibiotic - principally staphylococci. It is also used in structural studies of ribosome function
It inhibits elongation at the EFG mediated translocation step.
Binds EFG-GDP and prevents its release from the ribosome
Blocks A site for next round of aa incorporation.
What is puromycin.
Puromycin mimics an aminoacyl-tRNA because it resembles an aromatic amino acid linked to a sugar-base.
Treated by ribosome as if it were an incoming aminoacyl-tRNA.
The amino acids are normally incorporated at CCA tail of tRNA. Puromycin mimics the structure but has a stable NH group, the aromatic R-group makes it look like a tyrosine but most importantly it doesn’t have a tRNA.
The puromycin enters the A site but as it doesn’t have tRNA it doesn’t have the anticodon so doesn’t bind to mRNA. (bound to ribosome not mRNA) A peptide bond forms between the peptide and the puromycin and as it isn’t attached to the mRNA it can drift off. Suicide inhibitor as can’t be reused. The peptidyl-puromycin adduct is released in the form of a polypeptidyl-puromycin.
What is diphtheria toxin.
63kDA polypeptide produced by Corynebacterium diphtheriae which catalyses the ADP-ribosylation of eEF2 (NAD+ used as cofactor) blocking eukaryotic protein synthesis as blocks elongation factor. It can then be released and do the same thing in another cell.
Made of two subunit A and B (A enters cell whilst B opens the membrane).
What is ricin.
A lectin protein present as 5% of the dry mass of the seeds of castor bean plants ricinus communis
Very toxin by inhalation, injection or ingestion for humans:
* LD50 is 5-10 ug/kg via inhalation/injection by t 30-40 mg/kg orally.
* Ingestion of 5-20 castor beans lethal in adults.
Developed by several states as a biological warfare agent in the past and involved in several incidents such as the Georgi Markov assassination. There is some interest in potential therapeutic applications but process has been slow.
Ricin is a type II ribosome inactivating protein (RIP)
Ricin is initially synthesised as a prepro-polypeptide that contains both A and B chains. The two are then split by proteolysis but are still linked by as disulphide cysteine-cysteine bridge
Chain B is a lectin glycoprotein which binds galactose allowing it to enter the cell through the membrane and the endoplasmic reticulum.
Chain A is an RNA N-glycosidase that binds and de-purinates a specific adenine of the 28S rRNA. The adenine molecule becomes sandwich ed between tow tyrosine rings in the catalytic cleft of the enzyme and is then hydrolysed.
