Lecture 15 Flashcards
First step of translation (Activation of monomer)
Amino acid is adenylated with AMP moiety from ATP. Then, adenylated amino acid is attached to tRNA by amino acyl-tRNA synthase with high bond to form aminoacyl-tRNA (also called charged tRNA).
What does initiation of translation need?
Initiation requires initiator tRNA that has fMET in prokaryotes and Met(methionine) in eukaryotes, and AUG start codon.
Describe preparationg of starting tRNA in initiation of translation in prokaryotes
- Met is attached to initiator tRNA.
2. Met is then formylated by transformylase to form fMET-tRNA.
What is special about fMet-tRNA
Methionine attached to tRNA that is not the initiator tRNA cannot be formylated.
What is the formyl donor in initiation of translation?
N10-formyl-H4folate
How does 16S subunit of rRNA bind to mRNA for initiation?
There shine-dalgarno sequence (purine rich mRNA) that occurs 6-10 bases before AUG. 16S subunit contains complementary nt sequences to Shine Dalgarno seq which directs the binding and positioning of mRNA on ribosome.
Describe formation of initiation complex in prokaryotes (4)
- Small subunit binds to mRNA using Shine-Dalgarno seq on mRNA
- initiation factors attach to mRNA.
- Charged initiator tRNA loads onto AUG in the P site by IF2-GTP
- 50S subunit binds to form 70S complex. Energy required in this process is generated by hydrolysis of GTP and result in release of Initiation factors.
Describe elongation of translation in prokaryotes (5)
- Next aminoacyl tRNA is delivered to the empty A site by EF-Tu-GTP (Elongation factor)
- Then, GTP is hydrolyzed and EF released
- Then, energy from breakage of bond between carboxyl group of amino acid and the tRNA (tRNA charging) in P site form a peptide bond between 2 amino acids on A site, catalyzed by ribozyme (enzymatic activity of RNA portion of large subunit)
- EF-G-GTP translocates the ribosome 3 nucleotides along the mRNA. As a result deacylated tRNA (no aa) is shifted to E site and released while dipeptide-tRNA is shifted into P site.
- Steps are repeated until a termination codon is encountered in A site.
What is the name of EF-Tu-GTP in eukaryotes?
EF-1A
What is the name of EF-F-GTP in eukaryotes?
EF-2
What is Diptheria and what is the cause of the disease
It is acute infectious disease affecting the upper respiratory tract and occasionally the skin, caused by toxin A from C ulcerans.
Where is Toxin A from and what is its effect
Toxin A is produced by lysogenic bacteriophage (beta phage) which infects bacteria. It catalyzes the transfer of ADP-ribose to host cells EF-2 (same function as EF-F-GTP), inactivating it and preventing translocation of ribosome, thus inhibiting protein synthesis.
What is the treatment of toxin A?
Nicotinamide to reverse reaction + antibiotics
Which stop codon does Release factor 1 recognize in termination of translation?
UAA, UAG
Which stop codon does Release factor 2 recognize in termination of translation?
UAA, UGA
Role of release factor 3 in termination of translation
Bound to GTP, it carries out GTPase activity.
What happens in termination of translation
When stop codon is in A site, release factor enters the A site, triggering the peptidyl transferase to hydrolyse the ester bond between tRNA and completed polypeptide chain. Then, tRNA and ribosome subunits are recycled.
Role of polysome (polyribosome)
increase efficiency of protein synthesis as more than 1 ribosome can translate 1 mRNA.
In prokaryotes, transcription and translation are coupled?
Yes
What protein carries out protein folding during translation?
Chaperones
Mechanism of streptomycine
inhibit initiation of translation by binding to 30S subunit, distorting its structure, which prevent binding of fMET-tRNA to the P-site of ribosome. Thus, this inhibits initiation thus assembly of full ribosome.
What bacteria does streptomycine target?
gram ‘-‘ such as TB
Mechanism of tetracycline
Inhibits elongation by interacting with 30S and 40S small ribosome subunits blocking access& therefore binding of aminoacyl-tRNA to A site.
Advantage of tetracycline
Specific for prokaryotes because eukaryotic cells doesn’t let tetracycline diffuse into the cell.
Mechanism of Erythromycin
Inhibit elongation by binding irreversible to 50S ribosome (large subunit), blocking translocation by EF-G, thus preventing elongation.
Which bacteria does erythromycine target?
gram ‘+’
Disadvantage of erythromycine
Resistance can occur through plasmid controlled alteration of the binding site for erythromycine on the ribosome.
Mechanism of chloramphenicol
inhibit elongation by binding to 50S ribosome and inhibiting peptidyl transferase activity, preventing peptide bond formation, thus preventing elongation.
Disadvantage of chloramphenicol
it can cause serious haematological toxicity since it may inhibit eukaryotic mitochondrial ribosome, so it is not used in developed countries
Mechanism of cycloheximide
inhibit elongation by inhibiting 60S subunit in eukaryotes and inhibiting peptidyl transferase activity, thereby preventing peptide bond formation , preventing elongation.
*** 60S = Eukaryote so this is used only for research
Why is cycloheximide used for research?
Translation by mt ribosome is unaffected by cycloheximide, so to study mt translation, cycloheximide is used.
Mechanism of puromycin
Puromycine is analogue of phenylalanine attached to the tRNA. It has amide bond instead of ester linkage, thus once incorporated, ribosome (Peptidyl transferase) cannot break amide bond to release this tRNA, inhibiting elongation and causing premature chain termination.
Disadvantage of puromycin
active in both eukaryote and prokaryotes
Types of post-translational modification (6)
- Folding or formation of disulfide bridge
- Covalent alteration (phosphorylation, glycosylation, hydroxylation)
- Proteolytic processing such as trimming
- protein degradation (ubiquitination)
- Addition of prosthetic group (e.g. heme)
- Prenylation (eg. lipid anchoring, farnesylation)
Describe phophorylation as post translational modification
Transfer of phosphate group from ATP to amino acid side chain by kinase
- usually occurs on OH group of serine or threonine or less frequently tyrosine residue
e.g. insulin tyrosine kinase receptor
2 types of glycosylation
- O-linked glycosylation
2. N-linked glycosylation
What is O-linked glycosylation
Carbohydrate chains are attached to serine & threonine OH group. Usually occur after protein reach the golgi apparatus.
What is N-linked glycosylation
Carbohydrate chains attached to amide nitrogen of asparagine. Usually occurs in ER& Golgi. Catalyzed by glycosyltransferase
What is role of glycosylation?
Alters the properties of proteins, changing their stability, solubility and physical bulk. Also it act as a recognition signals that direct protein targeting to either plasma membrane or to organelle interiors or organelle membrane.
Also as a recognition signal, it influence cell to cell interaction.
Lipid anchoring as post-translational modification
Example : RAS protein
Farnesyl (15 carbon isoprenoid group) is attached to the cysteine of protein by farnesyltransferase.
Then, RAS is anchored to cytosolic face of cell membrane through farnesyl group.
What is proteolytic processing used for as post-translational modification?
zymogen activation to prevent premature activation of hydrolytic enzymes
Example of zymogen activation
Trpsinogen is cleaved into trypsin by enteropeptidase in small intestine (enteropeptidase expression happen only in small intesting to prevent autocatalytic pancreatitis).
Example of insulin as zymogen activation
Preproinsulin contains amino-terminal signal sequence that targets polypeptide chain to ER. Then, removal of signal sequence yields a second precursor, called proinsulin that has C-peptide. Then proinsulin is converted to insulin by proteolytic removal of C-peptide at Golgi body.
Ribosome configuration for prokaryotes and eukaryotes
Prokaryote : 50S+30S = 70S
Eukaryote : 60S + 40S = 80S
Difference in initiator tRNA between prokaryotes and eukaryotes
prokaryote : carry fMet
eukaryote : carry Met
Difference in setting correct reading frame in the beginning of translation between prokaryotes and eukaryotes
Prokaryote : Shine dalgarno seq
Eukaryote : scanning mechanism
Initiation factor of prokaryotes and eukaryotes
prokaryote : IF 1,2,3
Eukaryote : IF2 + additional factor
Elongation-aminoacyl tRNA delivery of prokaryotes and eukaryotes
prokaryote : EF-Tu-GTP
Eukaryote : EF-1-GTP
Elongation - translation factor of prokaryotes and eukaryotes
Prokaryote : EF-G-GTP
Eukaryote EF-2-GTP
