Protein Translation & Modification Flashcards
Where does trxn occur? Where does translation occur?
trxn= nucleus; translation= cytosol
-means the mRNA strand has to relocate
How does mRNA get into cytosol to be translated?
- via nuclear pore complex
- must be packaged in a specific manner, if incorrectly packaged will not be recognized by nuclear pore & won’t be able to move into cytosol
- movement of mRNA into cytosol IS A RATE LIMITING STEP
What are eukaryotic ribosomes? Where made? What is their role?
- individual subunits, 60s=large, 40s= small, together get 80s
- produced & assembled in nucleus, exported as separate subunits to cytosol
- factory responsible for translation
Roles of the ribosomal subunits?
- 40s (small) locates & bind mRNA, oversees tRNA binding to codons
- 60s (large) catalyzes peptide bond formation
- 80s (both) assembles upon initation of translation
what does “s” stand for in 80s vs 60s ribosomal subunits?
sedimentation coefficient
Prokaryotic ribosome structure?
Large (50s) subunit
small (30s) subunit
80s ribosomal structure during translation?
- three tRNA binding sites, one mRNA binding sites.
- right to left*
1) A: amino-acetyl-tRNA site binds incoming tRNA
2) P: peptide site: growing polypeptide chaine is attached
3) E: exit site where empty tRNA is released
4 steps of protein translation?
1) Preinitiation
2) Initiation
3) Elongation
4) Termination
What is the pre-initiation step in translation?
1) binding of methionine-tRNA & mRNA to 40s subunit
forms the pre-INITATION COMPLEX
What is the initiation step in protein translation?
1) binding of 60s subunits to pre-initation complex
2) scanning, recognizing initiating start codon AUG
What is the elongation step in translation?
1) new tRNA enters (decoding)
2) peptide bond forms (transpeptidation)
3) ribosomal subunit resets (translocation)
What is the termination step in translation?
1) stop codon recognition
2) ribosomal dislocation
3) terminal transferase activity c-terminal COOH
What are initiation & elongation factors?
- proteins that have roles in translation but aren’t associated w/ ribosome
- oncogenes & tumor supressor genes modify these factors to affect protein synthesis
- drugs can inhibit their acitivy to block translation
1) Initation factors role?
2) Elongation factors role?
1) involved in pre-initation/initation phase
2) assit in ribosomal peptide bond formation, translocation of mRNA, resetting ribosomes
eIF2A?
eIF2b?
- eukaryotic initiation factor *
1) drives MET-tRNA binding to 40s subunit via GDP–>GTP
2) exchanges eIF2A’s GDP–>GTP
makes pre-initation complex
Rate limiting step in the production of pre-initation complex?
binding of 5’CAP of the mRNA by initation factors which then help the mRNA find the preinitation complex
-because requires GTPase activity & IF/stabilization factors
What specifically happens during initation phase?
1) eIF4A unwinds mRNA, helps MET-tRNA find start codon
2) eIF5B helps large subunit bind, accelerates GTPase activity of 2A
3) GTP hydrolysis allows eIF2A to dissociate
Decoding step of elongation?
- new incoming tRNA sits in the A site catalyzed by EF1A
- GTP hydrolyzed when tRNA/codon pairing occurs
GTP hydrolysis mean?
GTP–>GDP via a GTPase
Transpeptidation step of elongation?
1) bond breaks between P-site tRNA and it’s polypeptide chain
2) new peptide bond formed w/ A-site tRNA
- catalyzed by 60s
3) rxt shifts 60s, moving tRNAs to E & P sites
Translocation step in elongation?
1) mRNA moves up 3 nucleotides
2) 40s resets w/ the already moved 60s to prepare new cycle
3) catalyzed by GTP bound Ef2
4) tRNA in E sites dissociates
What is the chain of termination?
1) termination
- stop codon reached
- no tRNA recognize termination codon, A site left open
2) release
- release factors bind A site
- peptidyltransferase ran hydrolyzes COOH end of polypeptide
- new protein released
3) disassembly
- mRNA, 40s, 60s dissociate
- requires GTP and RF’s (release factors)
What happens if pre-initiation, initiation, elongation & termination don’t happen correctly?
- if any part of process is completely blocked = DEATH
- if not completely blocked…just altered can result in increased/decrased translation
What are the most common errors in translation?
- synthesis of erroneous proteins (wrong aa added)
- premature abandonment of translation
- due to errors by the ribosomes
What affects the rate of translation?
-the regulation of the accessory proteins
initiation & elongation factors
How do signals altering eIFs lead to cancer?
- many growth factors cause phosphorylation of IFs, increasing their affinity for 5’cap, & increasing transaltion
- some tumor suppressors are phosphatases that dephosphorylate eIF & decrease translation, therefore decreases the amount of tumor suppressors
How control increasing or decreasing translation in regards to eIF-4E?
- phosphorylation by binding proteins INCREASES translation
- dephosphorylation DECREASES translation
Where do proteins destined for Plasma membrane & specific organelles go for processing?
- the proteins contain a signal sequence that targets the ER
- sorting sequences define end location
- additional proteins recognize the sequence & help w/ targeting
Importance of protein folding?
- need to be properly folded into functional structures
- proper folding is essential for proper modification, targeting & function
What are post-translational modifications? What determines which ones to apply to a protein?
- involve any change in the overall chemical nature of the protein after translation
- used to direct protein to it’s final destination
- based on specific cell type’s needs, environment, and interacting molecules
Why need post translation modifications?
- so that proteins can perform specific functions & go to correct final desitnation
- increases diversity & functionality of all proteins in cell (proteome)
Most of the modifications produced in the ER?
-are constitutive & remain until protein is degraded
Types of protein modifications?
-phosphoryaltion,N-linked glycosylation, disulfide bonds, lipidation, sulfating, ubiquitation, sumoylation
What is phosphorylation?
- a PTM
- reversible addition of a phosphate (PO4) group
- on serine, threonine, tyrosine
- promotes -activation/deactiviation of enzymes/receptors via conformational changes
- dynamic & regulated, involved in signal transduction
kinase vs phosphatase?
- kinase: adds phosphate group
- phosphatase: removes phosphate group
What is N-linked Glycosylation? Where occur?
- only in Eukaryotes
- covalent addition of sugars/carbs to ASN reside of protein
- occurs in ER at an Asn-X-Ser/Thr consensus sequence
- is a quality control mechanism
How N-linked glycosylation occur?
- occurs COTRANSLATIONALLY
- catalyzed by glycosyl transferases
- initial sugar added as one preformed unit
Why do we have glycosylation?
Essential for function of some proteins:
1) protects from proteases
2) can facilitate proper folding
3) can participate I cell adhesion/ligand recognition
4) regulated glycosylation can alter protein function
disulfide bond formation
- occurs in ER (oxidative env)
- formed between 2 cysteine
- sometimes required for protein folding, may require glycosylation to fold properly
- formed/broken by PDI
what is protein disulphide isomerase? relationship to disease?
- catalyzes formation & breakage of disulfide bonds
- via oxidation of cysteine
- checks for & corrects incorrect cys-cys bridges
- in the ER
Where disulfide bonds occur?
- oxidative env
- never in cytosol
- extracellualr faces of transmembrane proteins
PDI & neurodegeneration
- over production of free radicals inhibit PDI
- decreased levels of PDI result in increased protein misfolding
- been linked to neurodegenerative diseases
cytosolic lipid modification? (3)
- faciliatie membrane & protein-protein interactions
1) myristoylation
2) palmitoylation
3) prenylation
myristoylation
irreversible addition of myristic acid moiety to N terminus
-in signaling proteins
palmitoylation
reversible addition of palmitate to sulfate of internal cys residues
-cytosol & er
prenylation
-reversible addition of farnesyl or geranyl groups to C-term cysteines
Where/how/what is sulfating?
- PTM
- occurs in trans-golgi
- important for many extraceluuar metric protein & mucous
Ubiquitination, what does it do?
- addition of ubiquitin protein to lys of target proteins
- targets protein for degradation
- involved in cell cycle regulation, DNA repair & protein trafficking
What is sumoylation?
- reversible addition of SUMO to target proteins, affect protein function
- regulates nuclear-cytosol transport, cell cycle, protein stability & localization
- deSUMOylating enzymes reverse modification
What are the consequences of sumoylation?
- mutations in SUMOylation enzymes or protein targets leads to incorrect protein trafficking
- associated w/ neurodegenerative disorders
