1. Protein Synthesis (Translation)
Dr. Shumaila Asim Lecture # 8

2. Protein Structure Made up of amino acids
Polypeptide- string of amino acids
20 amino acids are arranged in different orders to make a variety of proteins
Assembled on a ribosome

3. Gene expression DNA  RNA  Protein DNA RNA Protein Replication
Initiation
Elongation
Processing
Export
Transcription
Degradation
Translation
Initiation
Elongation
Processing
Targeting
Degradation

4. Review DNA RNA Protein Gene 2 DNA molecule Gene 1 Gene 3 DNA strand
(template) 3¢ 5¢
TRANSCRIPTION
RNA
mRNA 5¢ 3¢
Codon
TRANSLATION
Protein
Protein
Amino acid

5. Review DNA RNA Protein Gene 2 DNA molecule Gene 1 Gene 3 DNA strand
(template) 3¢ 5¢
TRANSCRIPTION
RNA
mRNA 5¢ 3¢
Codon
TRANSLATION
Protein
Protein
Amino acid

6. Codon Table Genetic Code Second mRNA base First mRNA base (5¢ end)
Third mRNA base (3¢ end)

7. Codon Table Genetic Code Memorize Stop Codons Second mRNA base
First mRNA base (5¢ end)
Third mRNA base (3¢ end)

8. Transcription RNA forms base pairs with DNA
C-G
A-U
Primary transcript- length of RNA that results from the process of transcription

9. Translation
Second stage of protein production
mRNA is on a ribosome

10. Transcription vs. Translation Review
Process by which genetic information encoded in DNA is copied onto messenger RNA
Occurs in the nucleus
DNA mRNA
Translation
Process by which information encoded in mRNA is used to assemble a protein at a ribosome
Occurs on a Ribosome
mRNA protein

11. RNA RNA is single stranded, not double stranded like DNA
RNA is short, only 1 gene long, where DNA is very long and contains many genes
RNA uses the sugar ribose instead of deoxyribose in DNA
RNA uses the base uracil (U) instead of thymine (T) in DNA.

12. Transfer RNA
Consists of a single RNA strand that is only about 80 nucleotides long
Each carries a specific amino acid on one end and has an anticodon on the other end
A special group of enzymes pairs up the proper tRNA molecules with their corresponding amino acids.
tRNA brings the amino acids to the ribosomes,
Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3 end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.)
(a) 3 C A G U * 5
Amino acid
attachment site
Hydrogen
bonds
Anticodon
The “anticodon” is the 3 RNA bases that matches the 3 bases of the codon on the mRNA molecule

13. Transfer RNA 3 dimensional tRNA molecule is roughly “L” shaped 5 3 A
(b) Three-dimensional structure
Symbol used
in the book
Amino acid
attachment site
Hydrogen
bonds
Anticodon A G 5 3
(c)

14. Three different schematics
tRNA structure 3¢
Amino acid
attachment site 5¢
~ 80 nt long
Three different schematics
Hydrogen
bonds
Anticodon
Two-dimensional structure
Amino acid
attachment site 5¢ 3¢
Hydrogen
bonds 3¢ 5¢
Anticodon
Anticodon
Three-dimensional structure
Symbol used in this book

15. tRNA Transfer RNA Bound to one amino acid on one end
Anticodon on the other end complements mRNA codon

16. Ribosomes
2 subunits, separate in cytoplasm until they join to begin translation
Large
Small
Contain 3 binding sites E P A

17. Ribosomes Facilitate specific coupling of anticodons with codons
Ribosomal structure
Two ribosomal subunits (large and small)
Made of proteins (ribosomal proteins) and ribosomal RNA (rRNA)
Form binding sites for mRNA and aa-tRNA

18. Exit tunnel
Growing
polypeptide
tRNA
molecules
Large
subunit E P A
Small
subunit 5¢
mRNA 3¢
Computer model of functioning ribosome

19. 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

20. Mammalian example of 80S rRNA

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

22. 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

23. 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.

24. 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

25. 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

26. 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

27. 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.

28. Elongation

29. 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

30. 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).

31. 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.

32. 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

33. 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).

34. 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

35. Summary of transcription and translation
DNA
Stage mRNA is transcribed from a DNA template. 1
mRNA
RNA polymerase
Amino acid
TRANSLATION
Stage Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. 2
Enzyme
tRNA
Initiator
tRNA
Anticodon
Stage Initiation of polypeptide synthesis 3
Large ribosomal subunit
The mRNA, the first tRNA, and the ribosomal subunits come together.
Start Codon
Small ribosomal subunit
mRNA

36. New peptide bond forming
Growing
polypeptide
Stage Elongation 4
A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time.
Codons
mRNA
Polypeptide
Stage Termination 5
The ribosome recognizes a stop codon. The poly-peptide is terminated and released.
Stop
Codon

37. Posttranslational Modification
What is it ?
Addition of groups or deletion of parts to make a finished protein
What groups ? How much ? Where ?
- methyl
- acetyl
- glyco
- phospho

38. Purposes of post-translational modifications
Quality control in the cytoplasm
Quality control in the ER
Selective post-translational proteolysis
Glycosylation in the ER and beyond: N-linked vs. O-linked
Other post-translational modifications
Modifications that alter location:
Acylation: myristoylation, palmitoylation, prenylation
GPI anchor formation

39. 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

40. 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.

41. Post translational Modification
Examples:
Chromatin Structure/function - acetylation
Regulation of mitochondrial processes –
phosphorylation
Evade immune system – glycosylation
Gene regulation – glycosylation
Recognition - glycosylation

42. Protein Glycosylation Common in Eukaryotic Proteins

44. Co-translational protein folding
Fact: first ~30 amino acids of the polypeptide chain present within the ribosome is constrained (the N-terminus emerges first)
Assumption: as soon as the nascent chain is extruded, it will start to fold co-translationally (i.e., acquire secondary structures, super-secondary structures, domains) until the complete polypeptide is produced and extruded
folding
assembly

45. Questions (Molecular Biology)
Diagrammatic representation of a Nucleosome
Tabulate the major differences between prokaryotic and Eukaryotic replication
Comparison of replication and transcription in a tabular form
Tabulate the post transcriptional changes in mRNA, rRNA and tRNA.
Diagrammatic representation of the process of translation

46. Questions (Nucleic Acids &its Metabolism)
Synthesis of AMP and GMP from IMP with enzymes and coenzymes)
Catabolism of AMP to uric acid (with enzymes and coenzymes)
Catabolism of GMP to uric acid (with enzymes and coenzymes)
Write a note on disorders of purine related disorders
Draw the nuclei of Purines and Pyrimidine nitrogenous bases showing the sources of atoms
Write a note on disorders of pyrimidine related disorders