Protein Synthesis-- Translation from RNA to Protein Flashcards
Why study protein synthesis?
- last opportunity for regulating gene expression
- rapid response to stimuli (transcription sometimes takes too long b/c in the nucleus)
- many pharmaceutics impact translation (i.e. antibiotics)
STOP codons
UAA (U Are Annoying)
UGA (U Go Away)
UAG (U Are Gone)
START codon
AUG (Methionine)
Nonsense Mutiation
STOP speaking that nonsense
mutation creating stop codon
Key Players in Translation
- mRNA: the code
- tRNA: the adapter
- the ribosome: the enzyme
- *** Above ONLY NEEDED for protein synthesis **
- factors: the multitude of proteins that make the system work (increase efficiency, but are not necessary for occurrence)
5’ cap
- specialized nucleotide that is required for BINDING OF INITIATION FACTORS
- stability
5’ UTR
sequence between 5’ cap and the initiation codon
Coding Region
sequence that gets translated into protein
3’ UTR
- sequence between the stop codon and the poly(A) tail
- site of key regulatory sequences
poly(A) tail
untemplated polyadenylate sequence averaging ~200 nucleotides that protects mRNA from degradation and increases translational efficiency
5’ UTR
3’ UTR
5’ UTR: regulation; non-coding
3’ UTR: regulation; non-coding; stability
Aminoacylation
AA added to 3’
- enzymes (aminoacyl tRNA synthetases) recognize tRNAs and add appropriate AA
- requires ATP
- ** ACTIVATION; HIGH ENERGY BOND FORMED via AMP; AA attached to 3’ end ***
Translational Fidelity
Synthesis site vs Editing site
identifies anticodon loop (only proper loop will fit in enzyme; SPECIFICITY OF SHAPE AND CHARGE)
- synthesis site: v high affinity for proper AA; if incorrect cannot access site unless similar in size and structure
- editing site: v high affinity for INCORRECT AA; if enters editing site it will be cleaved off
Anticodon Loop
- recognizes codon
- aminoacylation adds proper AA to 3’ end of tRNA
- ATP driven
Ribosome = Ribozyme
rRNA is sufficient to carry out protein formation; it is an enzyme but poor efficiency w/o additional enzymes
Ribosome Binding Sites
A (aminoacyl): initial tRNA binding site for next codon
P (peptidyl): peptide bond formation
E (exit): tRNA release
Attach’ Put on bond; Exit
Translation Cycle
Initiation (Initiation Factor)
Elongation (Elongation Factor)
Termination (RELIEF Factor)
Initiation and Elongation occur at the same time with different ribosomes
Polyzome
mRNA + multiple ribosomes
mRNA ribosome complex
Initiation
- assembly of ribosome and mRNA
- positioning of the ribosome on the start codon (AUG)
Where does initiator tRNA enter at?
tRNA-MET enters at P site; all others enter at A site
** INITIATION tRNA DIFF THAN NORMAL MET tRNA (different one used during elongation) **
eIF4E
Initiation
binds to the 5’ cap; recruits eIF4G
eIF2
Initiation
binds and delivers initiator Met-tRNA (requires GTP)
eIF4G
Initiation
scaffold protein that binds eIF4E; required for assembly of pre-initiation complex
- bound at 5’ end and interacts with 3’ end creating circular mRNA allowing subunit to attach
Large subunit attachment
small subunit attaches post circularization of mRNA; once attached will scan for AUG codon; once reaches AUG large unit will come and attach
eIF2 release
(eIF2 binds and delivers intiation Met-tRNA to P site)
GTP hydrolysis cause eIF2 release
- once released large subunit attaches
Elongation
movement of the ribosome down the mRNA coordinated with aminoacyl tRNA delivery
rRNA site Occupation
at any given time only TWO SITES OCCUPIED
Initiation Steps Overview
1) formation of pre-initiation complex
2) Scanning to AUG codon
Elongation Steps Overview
1) delivery of aa-tRNA to A site and E site release
2) GTP hydrolysis and eEF1A release
3) eEF2 binding to catalyze translocation
4) GTP hydrolysis and eEF2 release; completion of cycle
eEF1A
Elongation
binds all cononical tRNAs (all except initiator and selenocysteine tRNA)
GTP hydrolysis is required
for release and promoting next step
eEF2
Elongation
- drags along large subunit; small unit follows; movement allows for elongation
- G-protein required for ribosome translocation
- if eEF2 is affected TRANSLOCATION CANNOT OCCUR, thus A site will never be empty and translation cannot proceed
“Proofreading” during Elongation
occurs in the ribosomal A-site where codon/anticodon pairs are “checked” by ribosome conformation
- incorrectly base-paired tRNAs preferentially dissociate
Termination
eRF2 binding and peptide hydrolysis
eRF1
- attaches and terminates protein synthesis
- recruited when STOP codon is recognized
- ** NO tRNA for STOP codons *** (tRAN mimetic and catalyzes release of completed peptide)
terminator tRNA
DOES NOT EXIST
Shine-Delgarno sequence
- initiation in prokaryotes
- upstream of start codon
- directly pairs to rRNA
Bacterial ribosomes allow for SELECTIVE inhibition by the ribosome inhibitor class of antibiotics
True
Mitochondrial Ribosomes
similar to bacterial ribosomes
- ribosome inhibitors have residual toxicity due to this similarity
Eukaryote vs Prokaryote Eukaryote - monocistronic mRNA - 40, 60, 80S ribosome - eIF4E - eIF2 - eIF4G - eEF1A - eEF2
Prokaryote
- polycistronic
- 30, 50, 70S ribosome
- eIF4E = no counterpart
- eIF2 = IF2
- eIF4G = no counterpart
- eEF1A = EFTu
- eEF2 = EFG
Hypoxia Regulation
- mTOR signaling pathway represses translation in response to hypoxia by regulating the function of eIF4E
- the mTOR pathway up-regulates during growth and down-regulates during stress
4EBPs
- increased affinity for eIF4E; binds and inhibits translation
- when phosphorylated they are released from eIF4E and translation inhibition is relieved
4EBP phosphorylation regulator
mTOR pathway; activation causes 4EBP phosphorylation and increases translation
mTOR repression
mTOR “normal”
mTOR high
repression: hypoxia; dephosphorylated, decreased translation
normal: normal phosphorylation, normal translation
high mTOR: response to growth stimulus or uncontrolled growth (cancer)
21st AA
Selenocysteine
- AUG codon (NOT stop); codon “re-coded” by SBP2
- cysteine with selenol instead of thiol
Hyperthyroidism
caused by selenocysteine deficiency
Diptheria toxin
shuts down translation by modifying the elongation factor responsible for translocation (eEF2)
Deiodinases
selenoproteins required for thyroid hormone maturation
DIO1-2-3
SBP2
protein required for recoding UGA to selenocyteine codon
EF1A and selenocysteine
EF1A delivers all tRNA to A site EXCEPT selenocysteine
-eEFSec only protein which can deliver selenocysteine to A site
SECIS
HOW mRNA KNOWS IF UGA IS STOP OR SELENOCYSTEINE
- when present UGA=> selenocysteine
- SBP2 binds exclusively to selenocysteine
