1. Protein Synthesis (Translation)

2. Proteins Synthesis Translation
Process by which ribosomes convert the information carried by mRNA to synthesize new proteins
mRNA is translated from 5 to 3-end producing a protein (peptide) synthesized from amino to carboxyl end

3. Requirements Ribosomes Amino acids mRNA tRNA Enzymes
Protein factors (initiation, elongation & release factors)
ATP & GTP as source of energy

4. Ribosome The ribosome has three binding sites for tRNA The P site
The A site
The E site

5. Schematic model showing binding sites on ribosome
P site (Peptidyl-tRNA
binding site)
A site (Aminoacyl-
tRNA binding site)
E site
(Exit site) E P A
Large
subunit
mRNA
binding site
Small
subunit

6. Mammalian example of 80S rRNA

7. Proteins Synthesis (Steps)
I. Activation of amino acids
(formation of amino-acyl-tRNAs or charged tRNA)
Amino-acyl-tRNA-synthetases recognize and catalyze the covalent attachment of a specific amino acid to CCA arm of tRNA
Amino acid + tRNA
Amino-acyl-tRNA
ATP
AMP + PPi  2Pi
First amino acid is always methionine (met-tRNA)

8. 1. Amino acid Aminoacyl-tRNA synthetase (enzyme) Pyrophosphate
Phosphates
tRNA
AMP
Aminoacyl tRNA
(an “activated
amino acid”)

9. II. Codon-anticodon recognition
Codon (on mRNA) & anti-codon (on tRNA) are anti-parallel to one-another

10. An initiation codon marks the start of an mRNA message
AUG = methionine
Start of genetic message
End
Figure 10.13A

11. Proteins Synthesis (Steps)
III. Initiation
Proper mRNA is selected by a ribosome ; ribosome attaches to it and reads the message from 5 to 3 end of mRNA
Ribosome dissociates into 40s & 60s sub-units
Formation of Pre-initiation complex (PIC) [40 s rRNA + met-tRNA + GTP + initiation factors (eIFs)]
mRNA binds to PIC forming 43 s initiation complex

12. More Initiation
The initiation process involves first joining the mRNA, the initiator methionine-tRNA, and the small ribosomal subunit. Several “initiation factors”--additional proteins--are also involved. The large ribosomal subunit then joins the complex.

13. Proteins Synthesis (Steps)
Binding of 43s (IC) with 60s ribosome forms 80s initiation complex, this complex contains P, A & E sites (“P” for peptidyl tRNA , “A” for new amino-acyl-tRNA & “E” for exit)
Starter / initiator codon (AUG) is identified by tRNA near the 5 end (cap end) of mRNA
This is determined by other sequences ,“Kozak consensus sequences” that surround “AUG”
met-tRNA occupies “P” site and is ready for elongation

14. mRNA, a specific tRNA, and the ribosome subunits assemble during initiation
Large ribosomal subunit
Initiator tRNA
P site
A site
Start codon
Small ribosomal subunit
mRNA 1 2

15. Elongation A cyclic process; one amino acid is added at a time
Sequence determined by the order of codons in mRNA
Requires “Elongation factors” (EFs)
Steps
Binding of amino acyl-tRNA to the “A” site
Peptide bond formation of this incoming amino acid with the carboxyl group (end) of met-tRNA (or growing peptide chain) at “P” site
Catalyzed by “Peptidyl-transferase” – a component of 28s rRNA (ribozyme) of the 60s ribosomal subunit

16. Elongation
This results in attachment of growing peptide to tRNA in the “A” site
Translocation – Now the ribosome advances three nucleotides (1 codon) towards 3-end of mRNA; results in
Movement of free tRNA (uncharged) from “P” to “E” site for release
EF2 displaces peptidyl-tRNA from “A” to “P” site
“A” site is now open for another amino-acyl-tRNA for another cycle of elongation

17. Termination
Elongation continues until a termination codon is encountered
Termination occurs when one of the three termination codons moves into “A” site
These codons are recognized by release factors
Release factors bind to and induce peptidyl transferase to release the polypeptide from tRNA

18. Termination
Three codons are called “stop codons”. They code for no amino acid, and all protein-coding regions end in a stop codon.
When the ribosome reaches a stop codon, there is no tRNA that binds to it. Instead, proteins called “release factors” bind, and cause the ribosome, the mRNA, and the new polypeptide to separate. The new polypeptide is completed.
Note that the mRNA continues on past the stop codon. The remaining portion is not translated: it is the 3’ untranslated region (3’ UTR).

19. 1. Recognition 3. Translocation 2. Peptide bond formation Amino end
of polypeptide E 3¢
mRNA P
site A
site
Ribosome ready for
next aminoacyl tRNA 5¢ 2
GTP
2 GDP E E P A P A
GDP
GTP
3. Translocation
2. Peptide bond
formation E P A

20. Ribosome translates 5’ to 3’ on mRNA.
Amino end
Growing polypeptide
Next amino acid
to be added to
polypeptide chain E
tRNA
mRNA 3¢
Codons 5¢
Schematic model with mRNA and tRNA
Ribosome translates 5’ to 3’ on mRNA.
Polypeptide chain grows amino end first, carboxyl end last.

21. 3¢ 3¢ 5¢ Release factor Stop codon (UAG, UAA, or UGA) Free polypeptide
When a ribosome reaches a stop
codon on mRNA, the A site of the
ribosome accepts a protein called
a release factor instead of tRNA. 3¢
The release factor hydrolyzes the
bond between the tRNA in the
P site and the last amino acid of the
polypeptide chain. The polypeptide
is thus freed from the ribosome.
The two ribosomal subunits
and the other components
of the assembly dissociate.

22. Polyribosomes
-a single mRNA (transcript) is translated by many ribosomes simultaneously
mRNA+ bound ribosomes= polyribosomes or polysome
Allows fast synthesis of many copies a polypeptide

23. Polyribosome or Polysome Completed polypeptides Growing polypeptides
Incoming
ribosomal
subunits
Polyribosome
Start of
mRNA
(5¢ end)
End of
mRNA
(3¢ end)
An mRNA molecule is generally translated simultaneously
by several ribosomes in clusters called polyribosomes.
Ribosomes
mRNA
0.1 mm
This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

24. RNA polymerase
DNA
mRNA
Polyribosome
Direction of
transcription
0.25 mm
RNA
polymerase
DNA
Polyribosome
Polypeptide
(amino end)
Ribosome
mRNA (5¢ end)

25. Targeting Polypeptides to Specific Locations
In eukaryotes, what are the two populations of ribosomes?
Free, soluble in cytosol
synthesize soluble proteins
Bound to rER
- synthesize secreted or membrane bound proteins
- tagged with signal peptide at amino end

26. Post-translation
The new polypeptide is now floating loose in the cytoplasm if translated by a free ribosome.
Polypeptides fold spontaneously into their active configuration, and they spontaneously join with other polypeptides to form the final proteins.
Often translation is not sufficient to make a functional protein, polypeptide chains are modified after translation
Sometimes other molecules are also attached to the polypeptides: sugars, lipids, phosphates, etc. All of these have special purposes for protein function.

27. Post-translational modifications
Trimming
Covalent modifications
Phosphorylation
Glycosylation
Hydroxylation
Other covalent modifications
Formation of disulfide links
Addition of carboxyl, acetyl group etc.
Addition of prosthetic group

28. Disulfide bonds form between cysteines
Disulfide isomerase works in the ER. In the cytosol most Cystines are in the reduced state partly because of active oxygen radical scavengers.
In the ER disulfide bond to another cystine within the target protein.