biology

1. Protein Synthesis
- mRNA is translated in 5'-3’protein from N-C-terminus).
 Proks mRNAs often have several coding regions(polycystronic).
- i.e., each coding region has its own initiation & termination codond/t PPchains.
 Each eukaryotic mRNA has only one coding region(monocistronic).
- In proks translation is coupled with transcription but not in euks.
- Translation has 4 steps in both proks & eukaryotic cells.
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2. Translation In Prokaryotes
1. Activation: binding of AA to its tRNA by aminoacyl-tRNA
synthetase:AA+ATP+tRNA=aminocyl-tRNA+PPi+AMP.
- The enzyme is specific to each tRNA & has editing activity.
2. Initiation:N-formyl-met is initiating AA on 5’-AUG-3’.
- 2 types of tRNAs in proks:tRNAfmet at the start codon & tRNAmet adds met at
the interior portion of a pp chain.
Initiation has 3 steps:
a) the 30s ribosomal subunit binds to IF-1&3.
- IF-3 prevents the reassociation of 30S with 50S.
- then the 30s binds to mRNA& initiating codon is guided to its correct position
by SD-sequence.
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3. Mechanism for Ribosomes to Recognize start codon
1. Shine-Dalgarno sequence(SD-sequence)
- Proposed by Australian scientists John Shine & Lynn Dalgarno.
- A purine-rich sequence of ntd bases in E.Coli.e.g.,5’-AGGAGGU-3’.
- located 6-10 bases upstream of the initiating AUG codon on the
mRNA(close to 5'-end).
- The 16S rRNA of the 30S subunit has a complimentary sequence
at its 3'-end(-Pyrimidine ACCUCCUUA-3'OH).
- SD-sequence doesn’t exist in Eukaryotes.
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4. Cont…
- Therefore, the 5' end of the mRNA & the 3'-end of the 16S r RNA
can form complementary base pairs brings 30S subunit on the mRNA
close to the initiating AUG Codon.
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5. Cont…
- The initiating codon 5’-AUG-3’ is positioned at the P-site where
fMet-tRNAfmet (The only aminoacyl-tRNA to bind the P-site) binds.
- IF-1 binds at A-site to prevent tRNA binding during initiation.
b) The complex of 30S,IF-3 & mRNA is joined by both GTP-IF-2 & the
initiating fMet-tRNAfmet
-The anticodon of this tRNA now pairs with the mRNA’s initiation codon.
c) The 2nd large complex binds with 50s subunit & GTP bound to IF-2.
dissociates to GDP+Pi w/c is released from the complex with all 3 IFs.
- then,50s & 30s associates to form functional 70s ribosome=initiation
complex(contain mRNA & initiating tRNA).
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Formation of the initiation complex (70s)
in bacteria in 3-steps.

8. 3.Elongation =Peptide bonds are formed
- requires the above initiation complex, amino acyl-tRNA & 3-EFs
(EF-Tu,EF-Ts,EF-G) & GTP. Has 3 repeating steps
a) Binding of an Incoming Aminoacyl-tRNA:charged-tRNA binds to a
complex of GTP-EF-TuaminoacyltRNA–EF-Tu–GTP complex binds to the
A site of the 70S initiation complex.
- GTP is hydrolyzed & EF-Tu–GDP complex is released from 70S ribosome.
- The EF-Tu–GTP complex is regenerated w/c involve EF-Ts & GTP.
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9. Cont…
b: Peptide Bond Formation: by peptidyl transferase(23rRNA)ribozyme.
- b/n the 2 AAs bound by their tRNAs to the A & P sites .
- transfer the initiating N-fmet from its tRNA to the NH2-group of the
2nd AA, in the A site.
- The α-NH2 group of the AA in the A-site acts as a nucleophile,
displacing the tRNA in the P-site to form the peptide bonddipep
tidyl-tRNA in the A site, then “uncharged” (deacylated) tRNAfMet
remains bound to the P site.
C) Translocation: the last step in elongation:the ribosome moves one codon
towards the 3’-end of mRNA.
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10. Cont…
- this mov’t shifts the anticodon of dipeptidyl-tRNA from the A to P-site;
also,the deacylated tRNA moves from P to E-site released.
- The 3rd Codon of mRNA now lies in the A site & the 2ndin P site.
- Movement of the ribosome along the mRNA requires EF-G(aka
translocase) & the energy by hydrolysis of another GTP.
- b/c EF-G can bind the A-site & displace the peptidyl-tRNA.
- The polypeptide remains attached to the tRNA of the most recent AA
to be inserted (EF-Tu has a proof reading activity).
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Elongation in 3 steps

12. 4.Termination
- Elongation adds 1AA at a time until it encounters the 1st stop Codon.
- Signaled by UAA,UAG or UGA on mRNA on the A-site.
- Bacteria have 3-termination or release factors(RF-1,2 & 3).
 Release Factors:
1) Hydrolyse the terminal peptidyl-tRNA bond;
2) release free ppchain & the last naked tRNA from P site
3) dissociation of the 70S ribosome30S &50Snew cycle.
 RF-1 recognizes UAG& UAA,and RF-2 recognizes UGA&UAA and bind.
- then they induce the peptidyl transferase to transfer the growing
Ppchain to water rather than another AA.
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13. Cont…
- The specific fun of RF-3 not well known,but it is thought to
release the ribosomal subunit.
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14. Antibiotics
1. Puromycin: has similar structure to the 3’-end of an aminoacyl-tRNA,
enabling it to bind to the ribosomal A site.
2. Tetracycline:blocks translation by binding to the A-site inhibits
aminoacyl-tRNA binding.
3. Chloramphenicol:inhibits bacterial,mitochondrial trans.by blocking
peptidyl transferase.
 Ricin:extremely toxic protein of the castor bean,inactivates the 60S of
euk ribosomes by depurinating a specific adenosine in 23S rRNA.
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15. Eukaryotic translation
1. Activation of amino acids:(formation of aminoacyl-tRNA):
- by aminoacyl-tRNA synthetase in cytosol not in ribosome.
- at least 20 d/t tRNAs & 20 d/t aminoacyl-tRNA synthetases
in a protein synthesizing system.
2. Initiation(2nd step):requires ribosome,mRNA,amino-acyl-tRNA,
GTP as energy source,IFs.
- IFs: facilitate the assembly of initiation complex.
- Eukaryotes:>10(eIF-1,2 etc).
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16. Cont…
 Initiation has 4 steps:
A) Dissociation of a ribosome:80S60S & 40S before initiation.
-then eIF-3& eIF-1A binds to 40S to
prevent reassociation of 60S & 40S).
- Also allows other IFs to bind with 40S80S initiation complex.
B) Formation of 43S pre-initiation complex(PIC).
- stabilized by association with eIF-3&eIF-1A.
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18. Cont…
- binding of GTP with elF-2eIf-2-GTP, w/c then binds to Met-
tRNA forms ternary complex.
- Met the 1st AA required to bind to the initiation codon AUG.
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19. Cont…
Initiation codon:-AUG is recognized by initiator-tRNA.
- facilitated by eIF-2-GTP other eIFs in Eukaryotes.
- Initiator-tRNA is recognized by eIF-2 then enter P-site directly.
- Initiator tRNA carries Met in euks(both fMet & Met are removed
from N-terminus before translation is completed).
- Then the large subunit joins the complex & forms functional ribosome
with charged initiator-tRNA in P-site & empty A-site.
- NB:The t-RNA binds with ribosome through the pseudouridine arm.
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20. Cont…
C. Formation of Initiation Complex
- binding of pre-initiation complex with mRNAinitiation complex (48s).
- in Euks mRNA is capped at 5’-end with Methyl-guanosine triphosphate.
- Methylation facilitates the binding & needs a cap binding protein
complex consisting of elF-4F, elF-4E and elF-4G + elF-4A.
- this protein complex binds the cap through elF-4E = 48s complex
w/c recognizes the 5’-cap (the rate limiting step in translation).
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21. Cont…
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-Then the 48s binds close to the 5’-cap & moves down the
mRNA until it encounters AUG w/c requires ATP.

22. 3.Elongation(3-steps)
- the addition of AAs to the carboxyl end of the growing chain.
- the ribosome moves from the 5’-end to3’-end of mRNA.
A. The binding of new aminoacyl-tRNA to ‘A’ site:
- in the 80S ribosomal initiation complex, the ‘P’ site is occupied
by met-tRNA & ‘A’ site is free.
- GTP bound EF-IA forms a ternary complex with the entering
aminoacyl-tRNA (A1)allows Amino acyl-tRNA to enter A-site.
- release of EF-1A-GDP+pi
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23. Cont…
B. Peptide bond formation.
- α-NH2 group of the new aminoacyl-tRNA(A1) in ‘A’ site combines
with the–COOH of Met-tRNA occupying the ‘P’ site.
- by Peptidyltransferase (component of 28S RNA of 60S subunit).
- since the AA on the aminoacyl-tRNA is already “activated”, the
rxn doesn’t require any further energyformation of a peptide
to the tRNA in the ‘A’ site. The peptide chain is at ‘A’ site &
naked tRNA at ‘P’ site.
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24. Cont…
C. Translocation process: by EF-2-GTP(translocase)
- EF-2-GTP binds to & displaces the peptide-tRNA from ‘A’ site‘P’
site & the deacylated tRNA is to ‘E-site’EF-2-GDP+pithe
ribosome moves forward by 1codon ,leaving the ‘A’ site free to
receive another ternary complex of a new AA elongation cycle.
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25. 4.Termination Process
- When the desired protein molecule is synthesized,a stop codon
appears in the A site of m-RNA(UAA,UAG & UGA).
- Euks have only 1 RF(eRF) that recognize all stop codons.
- no tRNA with an anticodon capable of recognizing stop codons.
- The binding of RFsdetachment of peptide from tRNA at the
P-site & nascent protein released from the ribosome.
- A 2ndfactor,eRF-3,act like prokaryotic RF-3.
- 80S ribosome now dissociates60S & 40Sw/c are then recycled.
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26. Polyribosomes/polysomes
- Many ribosomes w/c work on the same mRNA simultaneously.
- In euks,a single ribosome can synthesise 400 peptide bonds/min.
- each ribosome may be 80-100 ntds apart(minimum 35 ntds).
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27. Protein targeting(2-types)
- In eukaryotes translation occurs in cytosol,but many proteins work
out of the cell or within a specific organelles.
- These proteins contains AA sequence that direct their destination.
1.Co-translational Targeting:during translation
- have N-terminal hydrophobic sequencesdirects to RER.
2.Post-translational targeting:proteins contain short,basic,nuclear
localzing signaldirects to the nucleus.
- mitochondrial proteins:amphipathic a-helical sequencemitochondria.
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28. Regulation of translation
 Gene expression is regulated mainly at transcription level & also
during translation .
 Mechanism in Euks:by covalent modification of eIF-2.
- When eIF-2 is phosphorylatedinactiveno protein synthesis.
Co-& Posttranslational modification of PPchains.Include:
A) Trimming: cut part of a new protein & active form is released.
- site: some in ER,GA & some in secretory vesicles(insulin).
- zymogens active enzymes.
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29. Cont….
4. Other Covalent modifications:to modify protein’s function.
-e/g.,Extra COOH on Glu of Vit-K dependent clotting factors.
- Biotin w/c is bound to ε-amino group of Lys residues of biotin-
dependent enzymes that catalyze carboxylation rxns.
Protein folding
- Spontaneously or aided by chaperones(HSP70)functional.
Protein degradation: Alzheimer’s disease ?
- defective Proteins are tagged by ubiquitin in eukaryotes & lon
protein in prokaryotesdegradation.
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30. B) Covalent attachment:
1) Phosphorylation: at the-OH containing AAs of a protein.
- may ↑se or ↓se the activity of the protein.
- By protein kinases & may be reversed by protein phosphatases.
2. Glycosylation:Proteins w/c will be part of a plasma membrane.
- N-glycosylation = CHO + amide N-Asn of a protein in ER.
- O-glycosylation= CHO+ -OH group of S,T,OH-Lys in Golgi.
3. Hydroxylation:Proline & lysine residues of collagen chains in ER.
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31. Thank you!
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