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Translation- An overview
 By- Professor (Dr.) Namrata Chhabra
Biochemistry For Medics- Lecture Notes
www.namrata.co
03/12...
Translation-Introduction
 The pathway of protein synthesis is called
Translation because the ‘language’ of the
nucleotide...
Components required for
Translation-
 Amino acids
 Transfer RNA
 Messenger RNA
 Aminoacyl t RNA synthetase
 Functiona...
Steps of Protein Synthesis
The process of protein synthesis is divided into
3 stages-
- Initiation
- Elongation
- Terminat...
Initiation
 Initiation of the protein synthesis involves the
assembly of the components of the translation
system before ...
Ribosomes
 Ribosomes are large complexes of protein and
 r RNA. They consist of two subunits- one large
and one small wh...
Structure of Ribosome
A and P sites on Ribosome
The ribosome has two binding sites for t RNA molecules,
each of which extends over both subunits...
03/12/14 9Biochemistry For Medics
Transfer RNA
At least one specific type of t RNA is required
per amino acid.
Two sites are important, one Amino acid
attac...
Structure of Transfer RNA
03/12/14 11Biochemistry For Medics
Initiation-
The small ribosomal subunit binds to Initiation Factor 3
(IF3).
The small subunit/IF3 complex binds to the
mRNA. Specifically, it binds to the sequence
AGGAGG, known as the Shine-Delgarn...
Meanwhile, the fmet tRNA binds to Initiation
Factor 2 (IF2), which promotes binding of the
tRNA to the start codon.
03/12/...
The small subunit/IF3 complex scans along the mRNA until it
encounters the start codon. The tRNA/IF2 complex also binds
to...
At this point, the large ribosomal subunit joins in. A
molecule of GTP is hydrolyzed, and the initiation
factors are relea...
When the ribosome is assembled, two tRNA binding sites are
created; these are designated 'P' and 'A' (P stands for
peptidy...
When the second tRNA base pairs with the appropriate
codon in the mRNA, an enzyme called
peptidyl transferase catalyzes th...
Elongation-
At this point, the whole ribosome shifts over one codon.
This shift requires several elongation factors (not s...
The next tRNA base pairs with the next codon, and peptidyl
transferase catalyzes the formation of a peptide bond
between t...
Once again, the ribosome shifts over, so that the uncharged
tRNA is expelled, and the tRNA with the peptide chain
occupies...
Elongation (Contd.)
03/12/14 22Biochemistry For Medics
Termination-
When a termination codon enters the A site,
translation halts. This is because there is no tRNA
with an anti ...
The release factor causes the translation complex to
fall apart, and cleaves the polypeptide from the final
tRNA. The poly...
Termination
03/12/14 25Biochemistry For Medics
Eukaryotic Translation
Eukaryotic translation is very similar overall to prokaryotic translation.
There are a few notable ...
Inhibitors of protein
synthesis
 The tetracyclines (tetracycline, doxycycline,
demeclocycline, minocycline, etc.) block
b...
Inhibitors of protein
synthesis
 Choramphenicol inhibits prokaryotic
peptidyl Transferase
 Clindamycin and Erythromycin ...
Post Translational
Modifications
The newly synthesized protein is modified to become
functionally active. The various post...
Post Translational
Modifications
Trimming removes excess amino acids.
Phosphorylation may activate or inactivate the prote...
Translation: Summary of Key
Points
 Translation is the synthesis of a polypeptide using the information
encoded in an mRN...
Summary of Translation
03/12/14 32Biochemistry For Medics
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  1. 1. Translation- An overview  By- Professor (Dr.) Namrata Chhabra Biochemistry For Medics- Lecture Notes www.namrata.co 03/12/14 1Biochemistry For Medics
  2. 2. Translation-Introduction  The pathway of protein synthesis is called Translation because the ‘language’ of the nucleotide sequence on the mRNA is translated into the language of the amino acid sequence.  The m RNA is translated from its 5’end to its 3’end , producing a protein synthesized from its amino terminal end to its carboxyl terminal end. 03/12/14 2Biochemistry For Medics
  3. 3. Components required for Translation-  Amino acids  Transfer RNA  Messenger RNA  Aminoacyl t RNA synthetase  Functionally competent ribosomes  Protein factors  ATP and GTP as a source of energy 03/12/14 3Biochemistry For Medics
  4. 4. Steps of Protein Synthesis The process of protein synthesis is divided into 3 stages- - Initiation - Elongation - Termination 03/12/14 4Biochemistry For Medics
  5. 5. Initiation  Initiation of the protein synthesis involves the assembly of the components of the translation system before the peptide bond formation occurs. These components include-  Two ribosomal subunits  m RNA, Aminoacyl tRNA specified by the codons in the message ,GTP and initiation factors that facilitate the assembly of this initiation complex.  In prokaryotes three initiation factors are known (IF-1,IF-2 and IF-3) while in eukaryotes there are at least 9, designated as e IF, to indicate the eukaryotic origin. 03/12/14 5Biochemistry For Medics
  6. 6. Ribosomes  Ribosomes are large complexes of protein and  r RNA. They consist of two subunits- one large and one small whose relative sizes are generally given in terms of their sedimentation coefficients or S (Svedberg) values.  The S values are determined by the shape as well as by the molecular mass, their numerical values are strictly not additive.  The prokaryotic 50S and 30S ribosomal subunits together form a ribosome with an S value of 70. 03/12/14 6Biochemistry For Medics
  7. 7. Structure of Ribosome
  8. 8. A and P sites on Ribosome The ribosome has two binding sites for t RNA molecules, each of which extends over both subunits. Together they cover the neighboring codons.During translation , The A site binds an incoming Aminoacyl t RNA as directed by the Codon currently occupying the site. This Codon specifies the next amino acid to be added to the growing peptide chain. The P site codon is occupied by the peptidyl-t RNA. This t RNA carries the chain of amino acids that has already been synthesized. An E site is also there that is occupied by the empty t RNA that is about to exit the ribosome 03/12/14 8Biochemistry For Medics
  9. 9. 03/12/14 9Biochemistry For Medics
  10. 10. Transfer RNA At least one specific type of t RNA is required per amino acid. Two sites are important, one Amino acid attachment site the other is Anticodon site. Because of their ability to carry a specific amino acid and to recognize the codons for that amino acid , t RNA are called the adapter molecules. 03/12/14 10Biochemistry For Medics
  11. 11. Structure of Transfer RNA 03/12/14 11Biochemistry For Medics
  12. 12. Initiation- The small ribosomal subunit binds to Initiation Factor 3 (IF3).
  13. 13. The small subunit/IF3 complex binds to the mRNA. Specifically, it binds to the sequence AGGAGG, known as the Shine-Delgarno sequence, which is found in all prokaryotic mRNAs. 03/12/14 13Biochemistry For Medics
  14. 14. Meanwhile, the fmet tRNA binds to Initiation Factor 2 (IF2), which promotes binding of the tRNA to the start codon. 03/12/14 14Biochemistry For Medics
  15. 15. The small subunit/IF3 complex scans along the mRNA until it encounters the start codon. The tRNA/IF2 complex also binds to the start codon. This complex of the small ribosomal subunit, IF3, initiator tRNA, and IF2 is called the initiation complex. 03/12/14 15Biochemistry For Medics
  16. 16. At this point, the large ribosomal subunit joins in. A molecule of GTP is hydrolyzed, and the initiation factors are released. The ribosomal complex is now ready for protein synthesis. 03/12/14 16Biochemistry For Medics
  17. 17. When the ribosome is assembled, two tRNA binding sites are created; these are designated 'P' and 'A' (P stands for peptidyl, A stands for amino acyl). The initiator tRNA is in the P site, and the A site will be filled by the tRNA with the anticodon that is complementary to the codon next to the start. (In this case, it is the tRNA that binds proline.) 03/12/14 17Biochemistry For Medics
  18. 18. When the second tRNA base pairs with the appropriate codon in the mRNA, an enzyme called peptidyl transferase catalyzes the formation of a peptide bond between the two amino acids present (while breaking the bond between fmet and its tRNA).This activity is intrinsic to the 23S r RNA found in the large subunit. Since the r RNA catalyzes this process , it is referred to as the Ribozyme 03/12/14 18Biochemistry For Medics
  19. 19. Elongation- At this point, the whole ribosome shifts over one codon. This shift requires several elongation factors (not shown) and energy from the hydrolysis of GTP. The result of the shift is that the uncharged tRNA that was in the P site is ejected, and the tRNA that was in the A site is now in the P site. The A site is free to accept the tRNA molecule with the appropriate anticodon for the next codon in the mRNA. 03/12/14 19Biochemistry For Medics
  20. 20. The next tRNA base pairs with the next codon, and peptidyl transferase catalyzes the formation of a peptide bond between the new amino acid and the growing peptide chain. 03/12/14 20Biochemistry For Medics
  21. 21. Once again, the ribosome shifts over, so that the uncharged tRNA is expelled, and the tRNA with the peptide chain occupies the P site. (This is why this site is called the 'peptidyl' site - after the shift, it contains the tRNA with the growing peptide chain. The other site will accept a tRNA with an amino acid, hence the name 'aminoacyl' site) . The process of shifting and peptide bond formation continues over and over until a termination codon is encountered. The elongation process is fairly rapid, with prokaryotic ribosomes able to add 15 amino acids to the growing polypeptide every second. The process is also relatively error-free. Only one mistake is made every 10,000 amino acids. For large proteins of 1000 amino acids, that would mean one wrong amino acid in every 10 polypeptides. Elongation (Contd.) 03/12/14 21Biochemistry For Medics
  22. 22. Elongation (Contd.) 03/12/14 22Biochemistry For Medics
  23. 23. Termination- When a termination codon enters the A site, translation halts. This is because there is no tRNA with an anti codon that is complementary to any of the stop codons. 03/12/14 23Biochemistry For Medics
  24. 24. The release factor causes the translation complex to fall apart, and cleaves the polypeptide from the final tRNA. The polypeptide product is now free to function in the cell. The mRNA molecule is now available to be translated again. Very often, more than one ribosome will translate a single mRNA at the same time. One ribosome will initiate translation, and after it moves down the mRNA a bit, another ribosome will initiate, then another, and so on. The structure consisting of multiple ribosomes translating a single mRNA molecule is called a polysome . Eventually, the mRNA is degraded, and translation of that particular message will cease. 03/12/14 24Biochemistry For Medics
  25. 25. Termination 03/12/14 25Biochemistry For Medics
  26. 26. Eukaryotic Translation Eukaryotic translation is very similar overall to prokaryotic translation. There are a few notable differences, These include the followings: Eukaryotic mRNAs do not contain a Shine-Delgarno sequence. Instead, ribosomal subunits recognize and bind to the 5' cap of eukaryotic mRNAs. In other words, the 5' cap takes the place of the Shine-Delgarno sequence. Eukaryotes do not use formyl methionine as the first amino acid in every polypeptide; ordinary methionine is used. Eukaryotes do have a specific initiator tRNA, however. Eukaryotic translation involves many more protein factors than prokaryotic translation (For example, eukaryotic initiation involves at least 10 factors, instead of the 3 in prokaryotes.) 03/12/14 26Biochemistry For Medics
  27. 27. Inhibitors of protein synthesis  The tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline, etc.) block bacterial translation by binding reversibly to the 30S subunit and distorting it in such a way that the anticodons of the charged tRNAs cannot align properly with the codons of the mRNA.  Puromycin structurally binds to the amino acyl  t RNA and becomes incorporated into the growing peptide chain thus causing inhibition of the further elongation. 03/12/14 27Biochemistry For Medics
  28. 28. Inhibitors of protein synthesis  Choramphenicol inhibits prokaryotic peptidyl Transferase  Clindamycin and Erythromycin bind irreversibly to a site on the 50 s subunit of the bacterial ribosome thus inhibit translocation.  Diphtheria toxin inactivates the eukaryotic elongation factors thus prevent translocation. 03/12/14 28Biochemistry For Medics
  29. 29. Post Translational Modifications The newly synthesized protein is modified to become functionally active. The various post translational modifications are as follows- -Trimming -Covalent Alterations a)Phosphorylation b) Glycosylation c)Hydroxylation d)Gamma carboxylation e) Isoprenylation -Protein degradation 03/12/14 29Biochemistry For Medics
  30. 30. Post Translational Modifications Trimming removes excess amino acids. Phosphorylation may activate or inactivate the protein Glycosylation targets a protein to become a part of the plasma membrane , or lysosomes or be secreted out of the cell Hydroxylation such as seen in collagen is required for acquiring the three dimensional structure and for imparting strength Defective proteins or destined for turn over are marked for destruction by attachment of a Ubiquitin protein. Proteins marked in this way are degraded by a cellular component known as the Proteasome. 03/12/14 30Biochemistry For Medics
  31. 31. Translation: Summary of Key Points  Translation is the synthesis of a polypeptide using the information encoded in an mRNA molecule. The process involves mRNA, tRNA, and ribosomes.  tRNA has a unique structure that exposes an anticodon, which binds to codons in an mRNA, and an opposite end that binds to a specific amino acid. Binding of an amino acid to a tRNA is carried out by an enzyme called aminoacyl tRNA synthetase in a process called charging.  Translation consists of three basic steps: initiation, elongation, and termination. Initiation involves the formation of the ribosome/mRNA/initiator tRNA complex.  Elongation is the actual synthesis of the polypeptide chain, by formation of peptide bonds between amino acids.  Termination dissociates the translation complex and releases the finished polypeptide chain. Each of these steps requires the activity of a specific set of protein factors in addition to the ribosome, tRNA, and mRNA. 03/12/14 31Biochemistry For Medics
  32. 32. Summary of Translation 03/12/14 32Biochemistry For Medics
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