L4: Translation

Question 1

What is central dogma?

Answer 1

The two-step process (transcription and translation) by which the information in genes flows into proteins: DNA → RNA → protein.

Question 2

What is the genetic codon?

Answer 2

 Three adjacent nucleotides in the 5 ́-3 ́direction on mRNA constitute a genetic codon or triplet codon.

 One genetic codon codes for one amino acid.

Question 3

What are the characters of genetic codons?

Answer 3

 There are 20 amino acids

 Each amino acid has specific codons (1 or more)  Each codon consists of 3 nucleotides

 There are 64 codons (43):

 61 codons: sense codons codes for 20 aa

 3 codons: nonsense codons or termination codons (UAA, UAG, UGA)

 AUG (which encode for methionine) act as an initiation codon for translation

Question 4

What are the characteristics of the genetic code?

Answer 4

“CTSURN1”

1- Colinear: bases of the codon in mRNA are read from 5’ to 3’ end.

2- Triplet: Triplet sequence on mRNA that specifies certain a.a.

3- Specific: a specific codon always codes for the specific a.a.

4- Universal: It is the same for all species i.e. plants, animals.

5- Redundant (degenerate): a given a.a may have more than one codon that specifies the same a.a. They are different in the 3rd base.

6- Non-overlapping & commaless (Without interruption): read from a fixed starting point as a continuous sequence bases, taken 3 at a time without punctuation between the codons, The genetic codons should be read continuously without spacing or overlapping.

Question 5

What is the definition of translation?

Answer 5

The synthesis of protein using mRNA as the template, in other words, to translate the nucleotide sequence of mRNA into the amino acid sequence of protein according to the genetic code.

Question 6

What are the requirements for protein synthesis?

Answer 6

“mRNA+tRNA+Ribosomes+A.A+ATP+AATSE+Protein factors”

1) A ribosome: protein-synthesizing machinery.

2) mRNA: carries the information needed for arranging the amino acids in the proper order of the specific protein.

3) tRNA: which carries the amino acids to the proper place in the polypeptide chain. 4) Amino acids: the building units of the protein.

5) Aminoacyl-tRNA synthetase enzyme: which connects the amino acids to the specific carrier tRNA.

6) Protein factors: initiation factors (IF), elongation factors (EF) & releasing factors (RF).

7) A source of energy: in the form of ATP and GTP.

Question 7

What are the subunits of the ribosome?

Answer 7

❖ The prokaryotic 50S and 30S ribosomal subunits form a 70S ribosome.

❖ The eukaryotic 60S and 40S ribosomal subunits form an 80S ribosome.

Question 8

What are the binding sites of the ribosome?

Answer 8

❖ The ribosome has 2 binding sites for tRNA molecules, the A and P sites in addition to the E site.

❖ During translation:
 A site binds an incoming aminoacyl-tRNA.
 The P site codon is occupied by peptidyl-tRNA.
 This tRNA carries the polypeptide chain of amino acids that have already been synthesized.

Question 9

tRNA characters

Answer 9

 The tRNA carries amino acids during translation.

 There is at least one specific tRNA for each amino acid.

 Some amino acids (those having > one codon) may be carried by > one tRNA type.

 When a tRNA carries an amino acid it is said to be charged, and when an amino acid is carried by a tRNA it is said to be activated.

Question 10

What are the steps of translation?

Answer 10

1) Activation of aa: synthesis of aminoacyl-tRNA

2) Initiation: formation of the initiation complex

3) Elongation: polypeptide chain synthesis

4) Termination: release of the polypeptide chain

Question 11

Activation of amino acids

Answer 11

 Amino acids to be used in the synthesis of a protein must be at first activated [the amino acid binds to its specific tRNA by ester bond].

 The enzyme is responsible for charging tRNA by its specific A.A is known as aminoacyl tRNA synthetase (there are at least 20 different aminoacyl tRNA synthetases).

 2 high-energy bonds (from 1 ATP) are needed for a.a activation.

Question 12

Initiation of DNA translation

Answer 12

 Assembly of the translation machinery before peptide bond formation.

 Ribosome assembles with the other 2 types of RNAs forming the initiation complex:
1) The 2 subunits of the ribosome binds to the mRNA strand.
2) 1st tRNA binds to mRNA.

Question 13

Elongation of DNA translation

Answer 13

 Elongation is a cyclic process involving several steps and is catalyzed by elongation factors.

Question 14

What are the stages of elongation in DNA translation?

Answer 14

1) Binding of the new aminoacyl tRNA to A site:
 tRNA brings the correct new amino acid to A- Site

2) peptide bond formation:
 Formation of a peptide bond between the old and the newly added amino acid according to codon-anticodon recognition

3) Translocation:
 Release of tRNA from P-Site

Question 15

Binding of new amino acyl-tRNA to the A site

Answer 15

 Entry of a new amino acyl-tRNA to the empty A site on the ribosome requires proper codon recognition (acc. to the complementary bases of both codon and anticodon).

“One GTP is needed for this process”

Question 16

Peptide-bond formation

Answer 16

 The COOH of the aminoacyl-tRNA in the P site binds with NH2 of new aminoacyl-tRNA in the A site.

 This reaction is catalyzed by the peptidyl transferase enzyme present inside the 60s subunit.

Question 17

Translocation

Answer 17

 After the peptide bond formation, the ribosome moves 3 nucleotides towards the 3ʹ end of mRNA.

 This requires eEF2 and GTP.

 Results of translocation:
- Release of uncharged tRNA from E (exit) site.
- Transfer of the newly formed peptidyl-tRNA from A site to occupy P site.
- The A site becomes free (can be occupied by another new aminoacyl-tRNA according to the codon-anticodon recognition).

Question 18

What is the energy needed for the formation of one peptide bond?

Answer 18

For each new peptide bond formed 4 high energy phosphate bonds are cleaved:
 2 (from 1 ATP) for activation
 1 GTP for binding of aminoacyl-tRNA to A site
 1 GTP for translocation

Question 19

Termination of DNA translation

Answer 19

 Elongation of polypeptide chain continues until A site is occupied by one of the 3 non-sense codon (UAA, UAG, UGA), stop signal of translation.

 Releasing factors (RF) appear in A site and can recognize all three termination codons.

 RF + GTP + peptidyl transferase promotes the hydrolysis of the bond between the peptide chain and tRNA occupying the P site.

 This hydrolysis leads to:
- Release of both peptide and tRNA.
- Dissociation of 80s ribosomes into 40s and 60s subunit.

Question 20

Polyribosome

Answer 20

Because of the length of the nucleotide sequence of most mRNAs, more than one ribosome can translate the same mRNA at the same time.

Question 21

When do post-translational modifications take place?

Answer 21

 Many polypeptide chains are modified either while they are still in the ribosome or after their synthesis is completed.

Question 22

What does the process of post-translational modification include?

Answer 22

1) Folding

2) Trimming

3) Covalent modification

Question 23

Protein folding

Answer 23

Folding of polypeptide primary structure of protein molecule to get secondary, tertiary and quaternary structure if present to become functioning.

Question 24

What is trimming?

Answer 24

Removal of part of the peptide chain.

Question 25

What is the aim of trimming?

Answer 25

Conversion of inactive protein to an active one:

• many proteins are formed as large precursors molecules that are functionally inactive and part of the chain must be removed to release the active molecule.

Question 26

What are examples of trimming?

Answer 26

-proinsulin is converted into insulin.

-Zymogen of GIT: e.g. inactive pancreatic trypsinogen becomes active trypsin.

Question 27

Covalent modification

Answer 27

• Proteins may be activated or inactivated by covalent attachment of a variety of chemical groups e.g.
• Phosphorylation: addition of phosphate group to the protein
• Hydroxylation: addition of Hydroxyl group to the protein as in the maturation of collagen
• Carboxylation: addition of carboxylic group to the protein as in the activation of some clotting factors
• Glycosylation: addition of CHO part to the protein.B

Question 28

How can the translation process be regulated?

Answer 28

-At the level of initiation

-At the level of elongation

Question 29

Regulation at the level of initiation.

Answer 29

-by control activity of initiation factors (IF-2)

Example:
- Interferon (anti-viral drug) stimulates IF-2 phosphorylation → inhibits translation → to decrease viral protein synthesis Stop the virus from growing and dividing.

-increasing Heme globin synthesis by preventing phosphorylation of IF-2.

Question 30

Regulation of translation at the level of elongation

Answer 30

Diphtheria toxin inhibits elongation factor 2 (EF-2) → inhibits host protein synthesis.

Question 31

erythromycin

Answer 31

Some antibiotics as erythromycin inhibit the translocation step, so decrease bacterial protein synthesis.

Question 32

Many mechanisms that control protein synthesis explain the pathophysiology of some diseases:

Answer 32

Diphtheria toxin inhibits EF-2 → inhibition of protein synthesis → leads to cell death