1. Protein Metabolism
Protein Synthesis

2. Protein Synthesis: an overview
PS proceeds from N -terminus to C-terminus (amino to carboxyl)
Ribosomes:read mRNA in 5’ to 3’ direction
Polyribosomes (polysomes)
Max. density ~ 1 ribosome/80 nucleotides

3. Protein Synthesis: an overview
Chain elongation: links the growing chain to incoming tRNA’s AA residue
AA residues: added to C-terminus
Ribosome (prokaryote) has 3 tRNA binding sites
The P site: peptidyl site
Binds peptidyl –tRNA
Peptidyl-tRNA holds polypeptide
After peptide bond formation, binds deacylated tRNA
The A site: amino-acyl site
Binds incoming aminoacyl-tRNA

4. Protein Synthesis: an overview
Ribosome (prokaryote) has 3 tRNA binding sites
After peptide bond formation
Deacylated tRNA released from P site
Replaced in P site by newly formed peptidyl-tRNA
A site is vacated
The E site: exit site
Recent finding
Largely confined to 50S subunit
Deacylated tRNA dissociate from the ribosome

6. Protein Synthesis: an overview
Five (5) major stages of protein synthesis
1.Activation of Amino Acids
20 AA
20 aminoacyl-tRNA synthetases
20 or more tRNAs (min 32)
ATP
Mg2+

7. Protein Synthesis: an overview
Five (5) major stages of protein synthesis
2.Initiation
mRNA
N-Formylmethionyl-tRNA
Initiation codon in mRNA (AUG)
Ribosome: 30S and 50S subunit
Initiation factors: IF-1. IF-2, IF-3
GTP
Mg2+

8. Protein Synthesis: an overview
Five (5) major stages of protein synthesis
3.Elongation
Initiation complex (functional 70S ribosome)
aminoacyl-tRNAs specified by codons
Peptidyl transferases
Elongation factors: EF-Tu. EF-Ts, EF-G
GTP
Mg2+

9. Protein Synthesis: an overview
Five (5) major stages of protein synthesis
4. Termination and Release
Termination codon in mRNA
Polypeptide releasing factor: RF1, RF2, RF3
ATP
5. Folding and Processing
Enzymes (lots!)
Cofactors (lots!)

10. P S: stage one; activation of AA
Takes place in the cytosol
Each AA is attached to specific tRNA
ATP to AMP + Ppi
Catalyzed by MG 2+ -depending aminoacyl-tRNA synthetases
Aminoacylated tRNA is said to be charged

11. P S: stage two; Initiation
Initiation codon recognized by tRNAfmet
N-formylmethionine residue (fmet)
Special tRNA
Special aminoacyl-tRNA synthetase
First: bind tRNAfmet with Met
Then: N –formulates the Met residue
Note: proteins are post-translationally modified
Deformylation of fmet residue
Sometimes: removal of N-terminal Met

13. P S: stage two; Initiation
Initiation codon recognized by tRNAfmet
In eukaryotes:
All polypeptides synthesized by cytoplasmic ribosomes
Begin with Met residues (not fmet)
Have special initiating tRNA
Mitochondrial/chloroplast products are like polypeptides

14. P S: stage two; Initiation
The Shine-Dalgarno Sequence:
John Shine and Lynn Dalgarno in 1974
An initiating signal in mRNA
8-13 bp to the 5’ side of initiation codon
4-9 purine residues
Base pair with (antiparallel):
Complementary pyrimidine-rich sequence
Nearnear 3’ end of 16SrRNA on 30S subunit
mRNA-rRNA interactions
Sets mRNA in the correct position
Initiation of transcription

16. P S: stage two; Initiation
Three Stages: assemble Initiation Complex
Requires Initiation factors
Not permanently associated with ribosome
3 (in E. coli ): IF-1, IF-2, IF-3
Stage one (1):
30S subunit binds IF-3, IF-1
Prevents premature joining of LG and SM subunits
mRNA binds to 30S
Initiation codon (AUG) to P-site on 30S subunit
Guided by Shine-Delgarno sequence

18. P S: stage two; Initiation
Three Stages: assemble Initiation Complex
Stage two (2):
30S subunit with IF-3 with mRNA binds w/IF-2
IF-2 is already bound to:
GTP
fMet-tRNA fmet
Anticodon and codon pair

20. P S: stage two; Initiation
Three Stages: assemble Initiation Complex
Stage three (3):
Large complex formed in stage 2 combines with 50S subunit
GTP to GDP and Pi
IF-3, IF-1, IF-2 are released
IF-3 released before 50S attaches
irreversible

22. P S: stage two; Initiation
Three Stages: assemble Initiation Complex
At end of Initiation:
fMet-tRNA fmet with mRNA with Ribosome complex is formed
fMet-tRNA fmet in P site
A site ready
Eukaryotic Initiation is similar
More initiation factors
No Shine-Dalgarno
Cap is located; the 1st AUG downstream

26. P S: stage three; Elongation
3 stage cycle that is repeated
Adds AA to C-terminus
Up to 40 residues/sec
Elongation factors (EF)

27. P S: stage three; Elongation
Aminoacyl-tRNA binding
GTP with EF-Tu with aminoacyl-tRNA binds to ribosome
aminoacyl-tRNA: bound in codon-anticodon interaction at A site
GTP to GDP + Pi
EF-Tu to GDP + Pi are released
Regenerate GTP
EF-Tu with GDP + EF-T3 to EF-Tu with EF-Ts + GDP
EF-Tu with EF-Ts + GTP to EF-Tu with GTP + EF-Ts

29. P S: stage three; Elongation
Transpeptidation:
Peptide bond formation
Transfer of N-formylmethionyl group
From tRNA in P site
To amino group of 2nd AA in A site
Forms a dipeptidyl-tRNA in A site
tRNA fmet in P site
Peptidyl transferases
Catalyzes bond formation on LG subunit
Catalyzed by 23S rRNA (Harry Noller, 1992)

31. P S: stage three; Elongation
Translocation:
Ribosomes moves toward 3’ end by one codon
Dipeptidyl-tRNA moves to Psite
Deacylated- tRNA fmet released
New codon (3rd) into A site
Shift requires
EF-G (translocase)
GTP
Believed to be accompanied by 3-D changes in ribosome

33. P S: stage three; Elongation
Repeat elongation cycle
Need 2 GTP for each added AA residue
Protein chain always remains attached to a tRNA

34. P S: stage four; TERMINATION
Signaled by termination codon
When termination codon is in the A site
3 releasing factors
RF1, RF2, RF3
Hydrolysis of terminal peptidyl-tRNA bond
Release of protein and last tRNA
Dissociation of ribosome

36. P S: stage four; TERMINATION
When termination codon is in the A site
3 releasing factors
RF1: reacts to UAG, UAA
RF2: reacts to UGA, UAA
RF3: ?
RF bind at termination codon
Peptidyl transferase gives chain to H2O
Eukaryotes
One RF: eRF
Recognizes all 3 termination codons

38. P S: stage five; post-translational modification
To become mature, polypeptides must fold to native conformations
Disulfide bonds must form
Multisubunit proteins: subunits must combine
Must be modified by enzymes
Proteolytic cleavage
Most common P-T modification
Eg: all proteins have fMet residue removed
Eg: Conversion of trypsinogen to trypsin

39. P S: stage five; post-translational modification
Covalent modifications
E.g.:
Methylations
Hydroxylations
Etc
Changes in functional groups and radical groups