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Lecture 18:
Translation in prokaryotes
Readings (chapter 6)
Course 281
Lessons for life
AIMS
• Understand the process of translation in
prokaryotes.
• Understand the molecular requirements to
translate a prokaryotic mRNA into a protein.
• Understand the sequence of events in
prokaryotic translation.
Review
The ribosome revisited
Three sites in the two subunits are associated
with translation:
• A site (aminoacyl site): the site where the
aminoacyl tRNA binds.
• P site (peptidyl site): the site where the
peptide bond is formed between two amino
acids.
• E site (Exit site): the site where the tRNA
leaves the ribosome.
The ribosome revisited
Translation process
What are the stages of translation (protein
synthesis)?
(1) Initiation
(2) Elongation
(3) Termination
Translation initiation
Initiation involves all the steps before the formation
of the peptide bond between the first two amino
acids in the peptide chain.
What molecules involved in translation
initiation?
1. mRNA
2. Ribosome
3. Specific initiator tRNA (start codon!)
4. Initiation factors
5. Energy (GTP guanine triphosphate)
Translation initiation in bacteria
Simply ☺
1.The small ribosome finds the mRNA
2.Finding the start codon
3.Place the start tRNA (Met tRNA) in its correct
location
4.Assembly of the entire ribosome (large and
small) and the start tRNA (tRNA Met)
We need scientific details. Don’t we?
Translation initiation in bacteria
Translation initiation in bacteria starts with
(Finding the mRNA):
1.The interaction between the small ribosomal
subunit (30S) and two initiation factors (IF 1
and IF 3).
2.The complex (30S ribosomal subunit + IF1 +
IF 3) bind to the mRNA at a specific location.
Translation initiation in bacteria
Where in the mRNA this complex binds?
What is unique about the beginning of
mRNA?
Translation initiation in bacteria
The ribosome binding site in mRNA
1.The binding site in mRNA is not only the start
codon (AUG). WHY?
2.A sequences upstream of the start codon are
essential for specific binding of ribosome to the
correct location.
•The ribosome binding site (RBS) in
prokaryotic mRNA is called Shine-Dalgarno
sequence.
Translation initiation in bacteria
Shine-Dalgarno sequence
• 8-12 specific nucleotide sequence upstream of
the start codon (of each gene/transcript).
• The sequence interacts with the complementary
sequence in 16S rRNA in the small ribosomal
subunit.
• Interacts specifically with the small ribosomal
subunit 30S.
Translation initiation in bacteria
Shine-Dalgarno sequence
• This ensures specificity of where the ribosome
assembles and start translating.
• This sequence helps translating a polycistronic
transcript and each gene therein independently!
Translation initiation in bacteria
Before each protein coding gene in a polycistronic
transcript, a ribosome binding site exist.
Translation initiation in bacteria
Shine-Dalgarno sequence
Translation initiation in bacteria
1. The interaction between the small ribosomal
subunit (30S) and two initiation factors (IF 1
and IF 3).
2. The complex (30S ribosomal subunit + IF1 +
IF 3) bind to the mRNA at a specific location.
3. A special initiator tRNA binds to the 30S
ribosome and mRNA at the start codon.
Translation initiation in bacteria
What is the start codon?
What is the start codon tRNA?
Translation initiation in bacteria
Initiator tRNA in bacteria
1.The initiator tRNA in bacteria recognizes the
start codon with a complementary anti-codon
sequence of:
5’ CAU 3’
Isn’t the start codon AUG?
What is happening?
Translation initiation in bacteria
Initiator tRNA in bacteria
• The initiator tRNA carries a specific modified
amino acid called formyl-methoionine (fMet-
tRNA). It is a methoionine with a formyl group
added.
• When AUG is in the middle of a transcript
another tRNA is used. It is called Met-tRNA.
Translation initiation in bacteria
Initiator tRNA in bacteria
1.The initiator tRNA (fMet-tRNA) gets carried to
the complex (30S ribosome + IF1 + IF 3) by
initiation factor IF2 using GTP.
Translation initiation in bacteria
Initiator tRNA in bacteria
2.Initiator tRNA binds to a specific site in the
ribosome (P site).
Translation initiation in bacteria
Functions of translation initiation factors
• IF 1:
• Blocks the A site in the ribosome so that
only P site is available for initiator tRNA
is available to bind to.
Translation initiation in bacteria
Functions of translation initiation factors
• IF 2:
• Carries the initiator tRNA to the small
ribosomal subunit and places it in the P
site.
Translation initiation in bacteria
Functions of translation initiation factors
• IF 3:
• Binds to the mRNA in ribosomal binding
site.
• Prevent the binding of the 50S
ribosomal large subunit to the small
one.
Translation initiation in bacteria
1. The interaction between the small ribosomal
subunit (30S) and two initiation factors (IF 1
and IF 3).
2. The complex (30S ribosomal subunit + IF1 +
IF 3) bind to the mRNA at a specific location.
3. A special initiator tRNA binds to the 30S
ribosome and mRNA at the start codon.
4. The 50S ribosomal subunit binds to the (30S +
mRNA + fMet-tRNA) using GTP as a source of
energy.
Translation initiation in bacteria
What about IFs?
Translation initiation in bacteria
• The initiation factors (IF1 and IF3) gets released
and the resulting complex is called the initiation
complex.
Translation initiation in bacteria
• Initiation complex includes:
1. fMet-tRNA.
2. mRNA.
3. Small ribosome.
4. Large ribosome.
Translation initiation in bacteria
Summary
Translation elongation in bacteria
Elongation is adding more amino acids carried by
tRNA to Met (the start amino acid.
What are the steps in translation elongation?
1.Amino-acyl tRNA (charged tRNA) binds to the
ribosome’s A site.
2.Peptide bond forms.
3.Ribosome moves (translocate) one codon
downstream.
Translation elongation in bacteria
What is needed for elongation?
1.Charged tRNA
2.Elongation factors (EF)
3.GTP
Translation elongation in bacteria
Elongation process:
1.fMet tRNA is bound to the AUG codon at P site.
2.Next codon is positioned in the A site.
Translation elongation in bacteria
Elongation process:
3. Appropriate amino-acyl
tRNA binds to the A site.
4.The charged tRNA is
brought to the ribosome by
elongation factors (EF
and GTP).
Translation elongation in bacteria
Elongation process:
5.Two amino-acyl tRNAs
are in positions P and A
and a peptide bond is
formed between the two
amino acids.
6.The bond between the
amino acid and tRNA at P
site is broken.
Translation elongation in bacteria
Elongation process:
7. A peptide bond is formed
between the free amino acid
from the P site and the one
at the A site by:
Peptidyl Transferase
Translation elongation in bacteria
Elongation process:
8.When a peptide bond is
formed the free tRNA is in
site P and the tRNA at site
A has two amino acids.
9.Ribosome moves one
codon downstream (3’).
Translation elongation in bacteria
Elongation process:
10. Free tRNA moves to
the E site.
11. A new charged tRNA
gets to the A site and the
cycle repeats.
Peptide bond formation
Translation elongation in bacteria
Summary 1
Translation elongation in bacteria
Summary 2
Translation termination in bacteria
Termination is signaled
by a stop codon.
1.Stop codons DO NOT
code for amino acids and
thus DO NOT have tRNAs.
2.Release Factors (RF)
which looks like tRNA binds
to the A site.
3.Peptide is cleaved by
peptidyl transferase at the
P site.
Translation termination in bacteria
4. Amino acid chain is released.
5. tRNA at E site and P site are released.
Translation termination in bacteria
6. Ribosome two units break free from the mRNA
and RF is released.
Translation termination in bacteria
Summary
Stuff to know
HAGenetics.org
Translation initiation
Translation elongation
Translation termination
Start codon
Stop codons
AUG
A site
P site
E site
Amino-acyl site
Peptidyl site
Exit site
50S subunit
30S subunit
Ribosome
70S subunit
Initiation factors
IF 1
IF 2
IF 3 Shine-Dalgarno sequence
fMet-tRNA
Elongation factors
EF
Peptidyl transferase
Release factor
RF
Expectations
• You know how translation process occurs in
prokaryotes.
• You know how ribosome finds the correct
location to start translation.
• You know the molecules needed in every step
of translation.
• You the sequence of events in prokaryotic
translation.
For a smile

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281 lec18 prokaryotic_translation

  • 1. Lecture 18: Translation in prokaryotes Readings (chapter 6) Course 281
  • 3. AIMS • Understand the process of translation in prokaryotes. • Understand the molecular requirements to translate a prokaryotic mRNA into a protein. • Understand the sequence of events in prokaryotic translation.
  • 5. The ribosome revisited Three sites in the two subunits are associated with translation: • A site (aminoacyl site): the site where the aminoacyl tRNA binds. • P site (peptidyl site): the site where the peptide bond is formed between two amino acids. • E site (Exit site): the site where the tRNA leaves the ribosome.
  • 7. Translation process What are the stages of translation (protein synthesis)? (1) Initiation (2) Elongation (3) Termination
  • 8. Translation initiation Initiation involves all the steps before the formation of the peptide bond between the first two amino acids in the peptide chain. What molecules involved in translation initiation? 1. mRNA 2. Ribosome 3. Specific initiator tRNA (start codon!) 4. Initiation factors 5. Energy (GTP guanine triphosphate)
  • 9. Translation initiation in bacteria Simply ☺ 1.The small ribosome finds the mRNA 2.Finding the start codon 3.Place the start tRNA (Met tRNA) in its correct location 4.Assembly of the entire ribosome (large and small) and the start tRNA (tRNA Met) We need scientific details. Don’t we?
  • 10. Translation initiation in bacteria Translation initiation in bacteria starts with (Finding the mRNA): 1.The interaction between the small ribosomal subunit (30S) and two initiation factors (IF 1 and IF 3). 2.The complex (30S ribosomal subunit + IF1 + IF 3) bind to the mRNA at a specific location.
  • 11. Translation initiation in bacteria Where in the mRNA this complex binds? What is unique about the beginning of mRNA?
  • 12. Translation initiation in bacteria The ribosome binding site in mRNA 1.The binding site in mRNA is not only the start codon (AUG). WHY? 2.A sequences upstream of the start codon are essential for specific binding of ribosome to the correct location. •The ribosome binding site (RBS) in prokaryotic mRNA is called Shine-Dalgarno sequence.
  • 13. Translation initiation in bacteria Shine-Dalgarno sequence • 8-12 specific nucleotide sequence upstream of the start codon (of each gene/transcript). • The sequence interacts with the complementary sequence in 16S rRNA in the small ribosomal subunit. • Interacts specifically with the small ribosomal subunit 30S.
  • 14. Translation initiation in bacteria Shine-Dalgarno sequence • This ensures specificity of where the ribosome assembles and start translating. • This sequence helps translating a polycistronic transcript and each gene therein independently!
  • 15. Translation initiation in bacteria Before each protein coding gene in a polycistronic transcript, a ribosome binding site exist.
  • 16. Translation initiation in bacteria Shine-Dalgarno sequence
  • 17. Translation initiation in bacteria 1. The interaction between the small ribosomal subunit (30S) and two initiation factors (IF 1 and IF 3). 2. The complex (30S ribosomal subunit + IF1 + IF 3) bind to the mRNA at a specific location. 3. A special initiator tRNA binds to the 30S ribosome and mRNA at the start codon.
  • 18. Translation initiation in bacteria What is the start codon? What is the start codon tRNA?
  • 19. Translation initiation in bacteria Initiator tRNA in bacteria 1.The initiator tRNA in bacteria recognizes the start codon with a complementary anti-codon sequence of: 5’ CAU 3’ Isn’t the start codon AUG? What is happening?
  • 20. Translation initiation in bacteria Initiator tRNA in bacteria • The initiator tRNA carries a specific modified amino acid called formyl-methoionine (fMet- tRNA). It is a methoionine with a formyl group added. • When AUG is in the middle of a transcript another tRNA is used. It is called Met-tRNA.
  • 21. Translation initiation in bacteria Initiator tRNA in bacteria 1.The initiator tRNA (fMet-tRNA) gets carried to the complex (30S ribosome + IF1 + IF 3) by initiation factor IF2 using GTP.
  • 22. Translation initiation in bacteria Initiator tRNA in bacteria 2.Initiator tRNA binds to a specific site in the ribosome (P site).
  • 23. Translation initiation in bacteria Functions of translation initiation factors • IF 1: • Blocks the A site in the ribosome so that only P site is available for initiator tRNA is available to bind to.
  • 24. Translation initiation in bacteria Functions of translation initiation factors • IF 2: • Carries the initiator tRNA to the small ribosomal subunit and places it in the P site.
  • 25. Translation initiation in bacteria Functions of translation initiation factors • IF 3: • Binds to the mRNA in ribosomal binding site. • Prevent the binding of the 50S ribosomal large subunit to the small one.
  • 26. Translation initiation in bacteria 1. The interaction between the small ribosomal subunit (30S) and two initiation factors (IF 1 and IF 3). 2. The complex (30S ribosomal subunit + IF1 + IF 3) bind to the mRNA at a specific location. 3. A special initiator tRNA binds to the 30S ribosome and mRNA at the start codon. 4. The 50S ribosomal subunit binds to the (30S + mRNA + fMet-tRNA) using GTP as a source of energy.
  • 27. Translation initiation in bacteria What about IFs?
  • 28. Translation initiation in bacteria • The initiation factors (IF1 and IF3) gets released and the resulting complex is called the initiation complex.
  • 29. Translation initiation in bacteria • Initiation complex includes: 1. fMet-tRNA. 2. mRNA. 3. Small ribosome. 4. Large ribosome.
  • 30. Translation initiation in bacteria Summary
  • 31. Translation elongation in bacteria Elongation is adding more amino acids carried by tRNA to Met (the start amino acid. What are the steps in translation elongation? 1.Amino-acyl tRNA (charged tRNA) binds to the ribosome’s A site. 2.Peptide bond forms. 3.Ribosome moves (translocate) one codon downstream.
  • 32. Translation elongation in bacteria What is needed for elongation? 1.Charged tRNA 2.Elongation factors (EF) 3.GTP
  • 33. Translation elongation in bacteria Elongation process: 1.fMet tRNA is bound to the AUG codon at P site. 2.Next codon is positioned in the A site.
  • 34. Translation elongation in bacteria Elongation process: 3. Appropriate amino-acyl tRNA binds to the A site. 4.The charged tRNA is brought to the ribosome by elongation factors (EF and GTP).
  • 35. Translation elongation in bacteria Elongation process: 5.Two amino-acyl tRNAs are in positions P and A and a peptide bond is formed between the two amino acids. 6.The bond between the amino acid and tRNA at P site is broken.
  • 36. Translation elongation in bacteria Elongation process: 7. A peptide bond is formed between the free amino acid from the P site and the one at the A site by: Peptidyl Transferase
  • 37. Translation elongation in bacteria Elongation process: 8.When a peptide bond is formed the free tRNA is in site P and the tRNA at site A has two amino acids. 9.Ribosome moves one codon downstream (3’).
  • 38. Translation elongation in bacteria Elongation process: 10. Free tRNA moves to the E site. 11. A new charged tRNA gets to the A site and the cycle repeats.
  • 40. Translation elongation in bacteria Summary 1
  • 41. Translation elongation in bacteria Summary 2
  • 42. Translation termination in bacteria Termination is signaled by a stop codon. 1.Stop codons DO NOT code for amino acids and thus DO NOT have tRNAs. 2.Release Factors (RF) which looks like tRNA binds to the A site. 3.Peptide is cleaved by peptidyl transferase at the P site.
  • 43. Translation termination in bacteria 4. Amino acid chain is released. 5. tRNA at E site and P site are released.
  • 44. Translation termination in bacteria 6. Ribosome two units break free from the mRNA and RF is released.
  • 45. Translation termination in bacteria Summary
  • 46. Stuff to know HAGenetics.org Translation initiation Translation elongation Translation termination Start codon Stop codons AUG A site P site E site Amino-acyl site Peptidyl site Exit site 50S subunit 30S subunit Ribosome 70S subunit Initiation factors IF 1 IF 2 IF 3 Shine-Dalgarno sequence fMet-tRNA Elongation factors EF Peptidyl transferase Release factor RF
  • 47. Expectations • You know how translation process occurs in prokaryotes. • You know how ribosome finds the correct location to start translation. • You know the molecules needed in every step of translation. • You the sequence of events in prokaryotic translation.