1. Mohammad Hanafi, MBBS., dr., MS.
PROTEIN SYNTHESIS
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Mohammad Hanafi, MBBS., dr., MS.

2. Protein Synthesis The production (synthesis) of proteins. 3 phases:
1. Transcription
2. RNA processing
3. Translation
Remember: DNA  RNA  Protein

3. DNA  RNA  Protein Eukaryotic Cell DNA Pre-mRNA mRNA Ribosome Protein
Nuclear
membrane
Transcription
RNA Processing
Translation
DNA
Pre-mRNA
mRNA
Ribosome
Protein
Eukaryotic Cell

4. DNA  RNA  Protein Prokaryotic Cell DNA mRNA Ribosome Protein
Transcription
Translation
DNA
mRNA
Ribosome
Protein
Prokaryotic Cell

5. Question:
How does RNA (ribonucleic acid) differ from DNA (deoxyribonucleic acid)?

6. RNA differs from DNA 1. RNA has a sugar ribose
DNA has a sugar deoxyribose
2. RNA contains uracil (U)
DNA has thymine (T)
3. RNA molecule is single-stranded
DNA is double-stranded

7. 1. Transcription Eukaryotic Cell DNA Pre-mRNA mRNA Ribosome Protein
Nuclear
membrane
Transcription
RNA Processing
Translation
DNA
Pre-mRNA
mRNA
Ribosome
Protein
Eukaryotic Cell

8. 1. Transcription
The transfer of information in the nucleus from a DNA molecule to an RNA molecule.
Only 1 DNA strand serves as the template
Starts at promoter DNA (TATA box)
Ends at terminator DNA (stop)
When complete, pre-RNA molecule is released.

9. Question:
What is the enzyme responsible for the production of the RNA molecule?

10. Answer: RNA Polymerase
Separates the DNA molecule by breaking the H-bonds between the bases.
Then moves along one of the DNA strands and links RNA nucleotides together.

11. 1. Transcription
DNA
pre-mRNA
RNA Polymerase

12. Question: DNA 5’-GCGTATG-3’
What would be the complementary RNA strand for the following DNA sequence?
DNA 5’-GCGTATG-3’

13. Answer:
DNA 5’-GCGTATG-3’
RNA 3’-CGCAUAC-5’

14. 2. RNA Processing Eukaryotic Cell DNA Pre-mRNA mRNA Ribosome Protein
Nuclear
membrane
Transcription
RNA Processing
Translation
DNA
Pre-mRNA
mRNA
Ribosome
Protein
Eukaryotic Cell

15. 2. RNA Processing Maturation of pre-RNA molecules.
Also occurs in the nucleus.
Introns spliced out by splicesome-enzyme and exons come together.
End product is a mature RNA molecule that leaves the nucleus to the cytoplasm.

16. 2. RNA Processing pre-RNA molecule intron exon exon exon
splicesome
exon
Mature RNA molecule

17. Types of RNA Three types of RNA: A. messenger RNA (mRNA)
B. transfer RNA (tRNA)
C. ribosome RNA (rRNA)
Remember: all produced in the nucleus!

18. A. Messenger RNA (mRNA)
Carries the information for a specific protein.
Made up of 500 to 1000 nucleotides long.
Made up of codons (sequence of three bases: AUG - methionine).
Each codon, is specific for an amino acid.
4 nucleotides in mRNA so theoretically there are 43 (64) possible combinations of codons
Only 20 a.a.’s to encode

19. A. Messenger RNA (mRNA) Primary structure of a protein A U G C aa1 aa2
start
codon
codon 2
codon 3
codon 4
codon 5
codon 6
codon 7
codon 1
methionine
glycine
serine
isoleucine
alanine
stop
codon
protein
Primary structure of a protein
aa1
aa2
aa3
aa4
aa5
aa6
peptide bonds

21. Genetic Codes Codons are written 5’ to 3’
Codons for the same a.a. tend to have the nucleotides in 1st and 2nd positon
Raises the possibility of 3 different reading frames depending on the start
AUG is unique - encodes methionine
- acts as an initiation codon

22. B. Transfer RNA (tRNA) Made up of 75 to 80 nucleotides long.
Picks up the appropriate amino acid floating in the cytoplasm (amino acid activating enzyme)
Transports amino acids to the mRNA.
Have anticodons that are complementary to mRNA codons.
Recognizes the appropriate codons on the mRNA and bonds to them with H-bonds.

23. B. Transfer RNA (tRNA) methionine amino acid attachment siteU A C
anticodon
methionine
amino acid

25. tRNA Coupling
Aminoacyl-tRNA synthetase is required to catalyze the attachment of the specific a.a. to it’s associated tRNA

26. C. Ribosomal RNA (rRNA)
Made up of rRNA is 100 to 3000 nucleotides long.
Important structural component of a ribosome.
Associates with proteins to form ribosomes.

27. Ribosomes Large and small subunits.
Composed of rRNA (40%) and proteins (60%).
Both units come together and help bind the mRNA and tRNA.
Two sites for tRNA
a. P site (first and last tRNA will attach)
b. A site

28. Ribosomes
Large
subunit P
Site A
Site
mRNA A U G C
Small subunit

29. Ribosomes Protein formation requires orderly events to progress
Ribosomes possess binding sites to achieve this
mRNA binding site - holds mRNA strand in place
Aminoacyl-tRNA site (A-site) - holds one tRNA molecule
Peptidyl-tRNA site (P-site) - holds another tRNA molecule
Exit site (E-site) - holds a third tRNA molecule

30. 3. Translation Eukaryotic Cell DNA Pre-mRNA mRNA Ribosome Protein
Nuclear
membrane
Transcription
RNA Processing
Translation
DNA
Pre-mRNA
mRNA
Ribosome
Protein
Eukaryotic Cell

31. 3. Translation Synthesis of proteins in the cytoplasm
Involves the following:
1. mRNA (codons)
2. tRNA (anticodons)
3. rRNA
4. ribosomes
5. amino acids

32. 3. Translation Three parts: 1. initiation: start codon (AUG)
2. elongation:
3. termination: stop codon (UAG)
Let’s make a PROTEIN!!!!.

33. 3. Translation
Large
subunit P
Site A
Site
mRNA A U G C
Small subunit

34. Initiation mRNA Ribosome Initiator tRNA (fMet tRNA in prokaryotes)
Initiation-requirements:
mRNA
Ribosome
Initiator tRNA (fMet tRNA in prokaryotes)
3 Initiation factors (IF1, IF2, IF3)
Mg2+
GTP (guanosine triphosphate)

35. Initiation-steps (e.g., prokaryotes):
30S ribosome subunit + IFs/GTP bind to AUG start codon and Shine-Dalgarno sequence composed of 8-12 purine-rich nucleotides upstream (e.g., AGGAGG).
Shine-Dalgarno sequence is complementary to 3’ 16S rRNA.
Initiator tRNA (fMet tRNA) binds AUG (with 30S subunit). All new prokaryote proteins begin with fMet (later removed).
fMet = formylmethionine (Met modified by transformylase; AUG at all other codon positions simply codes for Met)
mRNA 5’-AUG-3’ start codon
tRNA 3’-UAC-5’ anti-codon
IF3 is removed and recycled.
IF1 & IF2 are released and GTP is hydrolysed, catalyzing the binding of 50S rRNA subunit.
Results in a 70S initiation complex (mRNA, 70S, fMet-tRNA)

37. Initiation G aa2 A U U A C aa1 A U G C U A C U U C G A codon 2-tRNA
anticodon A U G C U A C U U C G A
hydrogen
bonds
codon
mRNA

38. Elongation of a polypeptide:
Binding of the aminoacyl tRNA (charged tRNA) to the ribosome.
Formation of the peptide bond.
Translocation of the ribosome to the next codon.

39. 3-1. Binding of the aminoacyl tRNA to the ribosome.
Ribosomes have two sites, P site (5’) and A site (3’) relative to the mRNA.
Synthesis begins with fMet (prokaryotes) in the P site, and aa-tRNA hydrogen bonded to the AUG initiation codon.
Next codon to be translated (downstream) is in the A site.
Incoming aminoacyl-tRNA (aa-tRNA) bound to elongation factor EF-Tu + GTP binds to the A site.
Hydrolysis of GTP releases EF-Tu, which is recycled.
Another elongation factor, EF-Ts, removes GDP, and binds another EF-Tu + GTP to the next aa-tRNA.
Cycle repeats after peptide bond and translocation.

40. Elongation peptide bond G A aa3 aa1 aa2 U A C G A U A U G C U A C U U
3-tRNA G A
aa3
aa1
aa2
1-tRNA
2-tRNA
anticodon U A C G A U A U G C U A C U U C G A
hydrogen
bonds
codon
mRNA

41. Ribosomes move over one codon
aa1
peptide bond
3-tRNA G A
aa3
aa2
1-tRNA U A C
(leaves)
2-tRNA G A U A U G C U A C U U C G A
mRNA
Ribosomes move over one codon

42. peptide bonds G C U aa4 aa1 aa2 aa3 G A U G A A A U G C U A C U U C G
4-tRNA G C U
aa4
aa1
aa2
aa3
2-tRNA
3-tRNA G A U G A A A U G C U A C U U C G A A C U
mRNA

43. Ribosomes move over one codon
peptide bonds
4-tRNA G C U
aa4
aa1
aa2
aa3
2-tRNA G A U
(leaves)
3-tRNA G A A A U G C U A C U U C G A A C U
mRNA
Ribosomes move over one codon

44. peptide bonds U G A aa5 aa1 aa2 aa4 aa3 G A A G C U G C U A C U U C G
5-tRNA
aa5
aa1
aa2
aa4
aa3
3-tRNA
4-tRNA G A A G C U G C U A C U U C G A A C U
mRNA

45. Ribosomes move over one codon
peptide bonds U G A
5-tRNA
aa5
aa1
aa2
aa3
aa4
3-tRNA G A A
4-tRNA G C U G C U A C U U C G A A C U
mRNA
Ribosomes move over one codon

46. Termination aa5 aa4 aa3 primary structure of a protein aa2 aa1 A C U C
terminator
or stop
codon
200-tRNA A C U C A U G U U U A G
mRNA

47. End Product
The end products of protein synthesis is a primary structure of a protein.
A sequence of amino acid bonded together by peptide bonds.
aa1
aa2
aa3
aa4
aa5
aa200
aa199

48. Polyribosome
Groups of ribosomes reading same mRNA simultaneously producing many proteins (polypeptides).
incoming
large
subunit
small subunit
polypeptide
mRNA 1 2 3 4 5 6 7

49. Question:
The anticodon UAC belongs to a tRNA that recognizes and binds to a particular amino acid.
What would be the DNA base code for this amino acid?

50. Answer:
tRNA - UAC (anticodon)
mRNA - AUG (codon)
DNA - TAC

51. PROTEOME
The proteome is the final product of genome expression and constitute all the proteins present in a cell at a particular time, It is considered as the central link between the genome and the cell.

52. Protein Synthesis Inhibitor:
Many of the antibiotics utilized for the treatment of bacterial infections as well as certain toxins function through the inhibition of translation. Inhibition can be affected at all stages of translation from initiation to elongation to termination.

53. Protein Synthesis Inhibitor:
Several Antibiotic and Toxin inhibitors of Translation:
Chloramphenicol: inhibits prokaryotic peptidyl transferase
Cycloheximide: inhibits eukaryotic peptidyl transferase
Diptheria toxin catalyzes ADP-ribosylation of and inactivation of eEF-2
Erythromycin: inhibits prokaryotic translocation through the ribosome large subunit

54. Protein Synthesis Inhibitor:
Fusidic acid: similar to erythromycin only by preventing EF-G from dissociating from the large subunit
Neomycin: similar in activity to streptomycin
Puromycin: resembles an aminoacyl-tRNA, interferes with peptide transfer resulting in premature termination in both prokaryotes and eukaryotes
Ricin: found in castor beans, catalyzes cleavage of the eukaryotic large subunit Rrna
Streptomycin: inhibits prokaryotic peptide chain initiation, also induces mRNA misreading
Tetracycline: inhibits prokaryotic aminoacyl-tRNA binding to the ribosome small subunit

55. THE GENETIC CODE
Genetic code is degenerate, unambiguous, none overlapping
The genetic code consists of 64 triplets of nucleotides. These triplets are called codons.
Genetic code is required to account for all 20 amino acids found in proteins. A two-letter code would have only 42 = 16 codons, which is not enough to account for all 20 amino acids, whereas a three-letter code would give 43 = 64 codons.
The 64 codons fall into groups, the members of each group coding for the same amino acid.

56. THE GENETIC CODE
Degeneracy all amino acid are coded by two, three, four or six codons except tryptophan and methionine have just a single codon each.
The code also has four punctuation codons, which indicate the points within an mRNA where translation of the nucleotide sequence should start and finish.
The initiation codon is usually 5′-AUG-3′, which also specifies methionine (so most newly synthesized polypeptides start with methionine), with a few mRNAs other codons such as 5′-GUG-3′ and 5′-UUG-3′ are used.
The three termination codons are 5′-UAG-3′, 5′-UAA-3′ and 5′-UGA-3′; these are sometimes called amber, opal and ochre, respectively.

57. THE GENETIC CODE
The code is not uambiguous because a given codon designates only one amino acid.
One codon, AUG serves two related functions:
It signals the start of translation.
It codes for the incorporation of the amino acid methionine (Met) into the growing polypeptide chain.
The genetic code can be expressed as either RNA codons or DNA codons:

58. THE GENETIC CODE
RNA codons:
Occur in messenger RNA (mRNA) and are the codons that are actually read during the synthesis of polypeptides. But each mRNA molecule acquires its sequence of nucleotides by transcription from the corresponding gene.

59. THE GENETIC CODE
The DNA Codons: (genes at the level of DNA):
These are the codons as they are read on the sense (5' to 3') strand of DNA. Except that the nucleotide thymidine (T) is found in place of uridine (U), they read the same as RNA codons. However, mRNA is actually synthesized using the antisense strand of DNA (3' to 5') as the template.