Lecture 15 Flashcards
What are the requirements for translation?
- mRNA
- ribosomes
- Charged tRNA
- initiation factors
- GTP
Basic components of translation?
- activation of the monomer
- initiation
- elongation
- termination
- processing the polymer
How does tRNA become charged tRNA?
- enzyme bound amino acid- adenylate
- Formation of the aminoacyl-tRNA
reaction is driven by the hydrolysis of pyrophosphate
What is the first AA in prokaryotes and in mitochondria?
N-formylmethionine (fMet)
What is the role of fMET?
It allows for initiation
Prokaryotes have two tRNA’s for methionine?
- one allows for the formation of fMAT
2. allows for AUG codon
Two tRNA’s that recognize AUG in eukaryotes?
- The first codon also uses MET, and it has a special tRNA for this first codon (but the MET amino acid is not
formylated) . - Normal MET for internal codons
What is the purpose of the Shine Dalgarno sequence?
This sequence is purine rich
found in prokaryotes to allow for correct alignment of the AUG codon
How do eukaryotes align the mRNA with the ribosome?
lack Shine Dalgarno sequence
eukaryotic small ribosome binds close to the cap at the 5’ end, scans until it encounters the AUG start codon.
The steps to protein synthesis -initiation?
- Initiation factors (IFs) aid in the formation of the 30S initiation complex.
- The charged initiator tRNA is brought to the P site of the 30S subunit by IF-2-GTP.
- GTP on IF-2 is hydrolyzed and initiation factors are released when the 50S subunit arrives to form the
70S initiation complex
Protein synthesis- elongation
- Elongation factor EF-Tu-GTP brings the appropriately charged tRNA to the codon in the empty A site (decoding). GTP on EF-Tu is hydrolyzed.
- Peptidyltransferase activity of the 23S rRNA of 50S subunit catalyzes peptide bond formation, transferring
the initiating amino acid (or peptide chain) from the P site to the amino acid at the A site (transpeptidation).
protein synthesis - translocation
- EF-G-GTP facilitates movement of the ribosome three nucleotides along the mRNA in the 5’ to 3’
direction. - Steps are repeated until a termination codon is encountered at the A site.
Protein synthesis- termination
A termination codon is recognized by a release factor (RF-1 or RF-2), which results in release of the newly synthesized protein. GTP on RF-3 is hydrolyzed. The synthesizing complex dissociates.
In prokaryotes the mRNA is what?
polycistronic
contains multiple genes
What is the toxin Diphtheria toxin?
inactivation of EF-2 by ADP-ribosylation
this prevents translocation during protein synthesis
Streptomycin
Prevents assembly of ribosome (binds to 30s subunit)
Tetracycline
Block elongation by preventing aminoacyl-tRNA access to the A-site
Erythromycin
Binds to the 50S subunit of the complete (70S) ribosome
prevents ribosome translocation
Chloramphenicol
Inhibits peptidyl transferase activity in prokaryotes
At high levels, may inhibit mitochondrial translation
Cycloheximide
Inhibits eukaryotic peptidyl transferase activity
Puromycin
Causes premature termination of translation in both prokaryotes and eukaryotes
What are the 6 major post-translational modifications?
- protein folding
- covalent alterations
- proteolytic processing
- addition of prosthetic groups
- prenylation
- protein degradation
zymogen activation
Activation by enzymatic cleavage of peptide bonds of the zymogen molecule
nonactive to active
protein phosphorylation by a kinase
phosphate is transferred from ATP to AA side chain
occurs on OH groups
most common post-translational modification
What are the two types of glycosylation?
- N-linked- addition of N to asparagine
2. O-linked- sugar is attached to serine or theonine
Lipid anchoring?
The cell targets Ras protein to the cytosolic face
(inner leaflet) of the plasma membrane by a lipid
anchor mechanism - with the aid of farnesyl groups.
What protein is essential for proper insulin folding?
C-peptide
Proteolytic Processing of Insulin
- gene for insulin is transcribed into mRNA
- translation of that mRNA
- Translation of the polypeptide is directed into the lumen of the rER and forms “preproinsulin”
- The signal sequence is cleaved in the lumen of the rER – forms “proinsulin”
- Proinsulin is moved from the rER to the golgi, where it is cleaved to form insulin and C-peptide
- insulin and C-peptide go into secretory vesicles
- secreted by exocytosis
