Translation

1. TRANSLATION
By- Sanju Sah
St. Xavier’s college, maitighar, Kathmandu
Department of Microbiology
1

2. 2

3. Introduction
• Translation - conversion of mRNA base sequences into protein
amino acid sequences which involves a step wise reading of the
message present in mRNA base sequences as codons and
translating it into the amino acid sequences of protein.
• The translation of mRNA messages requires the following two
RNA species.
1. Transfer RNA(tRNA)
2. Ribosomal RNA(rRNA)
3

4. Different RNAs
1. Messenger RNA(mRNA)
 Messenger RNA is a single stranded base-for-base
complementary copy of one DNA strand of the coding
sequence of a gene and provides the information for amino
acid sequence of the polypeptide specified by that gene.
 It functions as a template for synthesis of protein.
 Least stable type of RNA.
 5-10% of the total cellular RNA.
 In Eukaryotes mRNA has certain modification after its
synthesis, considered as post transcriptional modification.
4

5. Polycistronic and monocistronic mRNA
• Generally, a single prokaryotic mRNA codes for more than one
poylpeptide, such an mRNA is called as polycistronic mRNA.
• Some correspond to a single gene known as monocistronic mRNA.
• All eukaryotic mRNAs are monocistronic.
• It contains three regions:
• coding region, 5’ leader region and 3’ trailer region.
• The coding region usually begins with AUG codon and ends with a
termination codons, the codons in between coding for the different amino
acids of the concerned polypeptide.
• The leader sequence is present at the 5’end before the AUG codon and is
not translated.
• The trailer sequence is located beyond the termination codon of the last
coding region which is also not translated. 5

6. 2. Transfer RNA (tRNA)
 Comprises about 10-15% of total cellular RNA.
 tRNA is transcribed in nucleus and must enter the cytoplasm.
 Each tRNA molecule links to a particular mRNA codon with a
particular amino acid.
 Size of the tRNA varies from 74-95 nucleotides.
 All tRNA molecules have a proportion of unusual bases;
dihydrouridine(Dih U), pseudouridine, inosine (I),
methylated bases and thymine(T).
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7. Transfer RNA:
adaptor between mRNA and amino acids
anticodon
codon
7

8. Sequence of tRNAala determined by Robert Holley in 1965.
Deduced “cloverleaf” structure
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9. Common features
1. All single-stranded nucleotide
chains of 73-93 ribonucleotides
2. Contain many unusual bases
3. The 5’ end is always
phosphorylated and usually a
pG
4. The 3’ end is always a -CCA-OH
5. Extensive base pairing,
highly conserved 3-D structure
Over 100 tRNA molecules have been sequenced
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10. 6. Anticodon loop contains 7
bases:
• pyr-pyr-X-Y-Z- modified purine-
variable
• Out of these 7 unpaired
nucleotides the middle three
form anticodon.
• Anticodon recognizes and
codon of mRNA and binds to it.
Over 100 tRNA molecules have been sequenced
10

11. Alexander Rich and Aaron Klug determined the 3-D structure of
yeast Phe-tRNA by X-Ray crystallography in 1974
3-D Structure of tRNAs
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12. 3. Ribosomal RNA( rRNA)
• Ribosomal RNA(rRNA) occurs in association with proteins
called ribosomes where amino acids are joined to form
protein primary structure - polypeptide
• Ribosomes of eukaryotes and prokaryotes differ in size and
other details.
• eukaryotic ribosomes - smaller 40S subunit has a single 18S
rRNA (1874 bases) and larger 60S subunit has one molecule
each of 28S (4718 bases), 5.8S (160 bases) and 5S (120 bases)
rRNA .
• The two subunits of ribosome dissociate in low magnesium
(Mg⁺⁺) concentration and re-associate in high Mg⁺⁺
concentration. 12

13. • Furthermore, each ribosome dissociates into the two subunits
every time it finishes reading an mRNA molecule.
• The two subunits reunite, at random, to produce complete
ribosomes once the smaller subunit attaches itself to he
initiation site of an mRNA.
• The larger subunit of ribosome carries the newly synthesized
protein, while the smaller subunit is associated with the
mRNA.
13

14. Ribosome
• Made of protein and rRNA
• has a large and small subunit
• has three binding sites for tRNA on its surface
• has one binding site for mRNA
• Facilitates codon and anticodon bonding
• Components of ribosome are made in the nucleus and
exported to the cytoplasm where they join to form one
functional unit.
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15. • The three tRNA binding sited are:
1. A site: holds tRNA that is carrying the next amino acid to be
added
2. P site: holds tRNA that is carrying the growing polypeptide
chain
3. E site: where discharged tRNA leave the ribosome.
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16. The Ribosome
Prokaryotic
Eukaryotic
30S and 50S subunit
40S and 60S subunit
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17. The Prokaryotic Ribosome
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18. 18
The Eukaryotic Ribosome

19. Genetic Code
• Genetic code - a language
that correspond between a
sequence of triplet
nucleotide bases of mRNA
and the sequence of
amino acid they specify.
• Each individual word is
composed of three
nucleotide bases which are
referred as codons.
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20. Information in DNA to Protein
• The DNA is first transcribed into mRNA inside the nucleus.
• mRNA then moves outside the nucleus to the ribosomes (rRNA),
where protein synthesis takes place.
• Ribosomes move along the mRNA strand, reading three
nucleotides at a time.
• The genetic code is also read by transfer RNAs (tRNA). Each tRNA
has an anticodon that is complementary to the codon in the
mRNA which then translate the codon into aminoacid.
• due to wobble base pairing the cell manages with less tRNAs than
would be expected from the number of anticodons required to
match the codons in the code table.
• Each tRNA carry the amino acid specified by the codon and joins
the amino acids together to make a polypeptide, which later folds
into protein.
20

21. features of genetic code
1. Triplet nature
The four nucleotide bases (A,G,C and U) in mRNA are used to
produce the three base codons.
64 combinations
• There are 64 codons in total including 61 sense codons (codons
that specify amino acid) and 3 non sense/stop codons (codon
that do not specify amino acid/signal termination).
• There are 61 codons that code for the 20 amino acids, and since
each codon code for only one amino acids this means that,
there are more than one code for the same amino acid. These
type of codons are also called as synonymous codon.
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22. 2. Commaless code
• the message is read in a continuing sequence of nucleotide
triplets until a translation stop codon is reached
3. Nonoverlapping code
• The codon is read in mRNA in a contiguous fashion. A
nucleotide that forms part of a triplet never forms part of
the next triplet
Each triplet is read from 5’ to 3’ direction.
5’ AUGUCUCCA 3’
5’ -AUG-UCU-CCA-3’ : Methionine, Serine, Proline
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23. 4. Degeneracy
• Most of the codons except methionine (AUG) and trytophan
(UGG) can decode the same amino acid , hence the genetic
code is degenerate.
• Most of this degeneracy involves the third nucleotide of a
codon. For e.g. threonine is coded by four codons ACU, ACC,
ACA and ACG.
5. Unambiguity
One codon codes for only one amino acid, hence, it is
unambiguous and specific.
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24. 6. Universality of code
• The genetic code is largely universal for all living organisms .
• few exception are found in mitochondria.
• Where, AUA code for Methionine and UGA, one of the termination
codons, code for tryptophan in yeast mitochondria.
• AGA and AGG code for Arginine in cytoplasm but in mitochondria
they are termination codons.
7. Terminating Codon/ Non Sense Codon
• There are 3 codons out of 64 in genetic code which do not encode
for amino acid.
• These are called termination codons or stop codons or nonsense
codons.
• The stop codons are UAA, UAG and UGA. The ribosome pauses and
falls off the mRNA .
8. Initiator Codon
• AUG is the initiator codon in majority of proteins 24

25. One tRNA can recognize more than one codon: Wobble hypothesis
1. The first two bases of an mRNA codon always
form strong Watson-Crick base pairs with the
corresponding bases of the tRNA anticodon
and confer most of the coding specificity.
2. The first base of anticodon determines the
number of codons recognized by the tRNA. For
e.g: When the first base of the anticodon is C or
A only one codon is recognized by that tRNA.
When the first base is U or G, two different
codons may be read.
3. When an amino acid is specified by several
different codons, the codons that differ in
either of the first two bases require different
tRNAs.
4. A minimum of 32 tRNAs are required to
translate all 61 codons (31 to encode the amino
acids and 1 for initiation). 25

26. 26
Polypeptide synthesis - Translation

27. Steps in Translation Process
Activation of Amino acid / Charging of t-RNA
Initiation
Elongation
Termination
Post translational modifications
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28.  Aminoacyl-tRNA synthetase esterify the 20 amino acids to their
corresponding tRNAs at the expense of ATP energy, using Mg ++ .
 When attached to their specific amino acid, the tRNAs are said to
be charged.
 Aminoacyl-tRNA synthetases accomplishes two things:
1. Activates the amino acid for polymerization
2. tRNA confers specificity by pairing with the mRNA codon
Stage 1: Amino acid activation
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29. Stage 2 : Initiation ( specific amino acid initiates
protein synthesis)
• Protein synthesis begins at the amino-terminal end and
proceeds by the stepwise addition of amino acids to the carboxyl-
terminal end of the growing polypeptide
• There are 2 methionyl tRNAs in prokaryotic organisms :
1. tRNAf or tRNAfmet for formyl-methionine
2. tRNAm or tRNAmet for methionine
• There is only one methionine aminoacyl tRNA synthetase
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30. • N-formylmethionyl tRNA fMet (fMet-tRNAfMet) arrives at the
ribosome.
• Addition of N-formyl group to the amino group of methionine
by the transformylase prevents fMet from entering interior
positions in a polypeptide while also allowing fMet-tRNAfMet
to be bound at specific ribosomal initiation P site that accepts
neither Met-tRNAMet nor any other aminoacyl-tRNA.
30

31. Stage 3: peptide bonds are formed in the elongation
stage
• The third stage of protein synthesis is elongation which
requires:
1. Initiation complex
2. Aminoacyl-tRNAs
3. A set of three soluble cytosolic proteins called the
elongation factors( EF-Tu, EF-Ts and EF-G) and
4. GTP
Cells use the three steps to add each amino acid residues, and the
steps are repeated as many times as there are residues to be
added.
31

32. Stage 4: Termination of polypeptide synthesis
• Elongation continues until the ribosome adds the last amino
acid coded by the mRNA
• Termination, the fourth stage of polypeptide synthesis, is
signaled by the presence of one of three termination codons in
the mRNA (UAA, UAG, UGA) immediately following the final
coded amino acid.
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33. tRNA charging by aminoacyl-tRNA synthetases
33
Stage 1: Amino acid activation

34. specificity
A specific N-formyl-
methionine tRNA
34
Stage 2 : Initiation ( specific amino acid initiates
protein synthesis)

35. ribosome distinguish start AUG and internal AUG?
Shine-Delgarno Sequence
prokaryotic translation start?
AUG is the Start Codon
35

36. 30S ribosomal subunit
Shine-Dalgarno Sequence Recognized?
The sequence of the 16 S rRNA features a short region of
complementarity with the Shine/Delgarno sequence.
36

37. • The initiation of polypeptide synthesis in bacteria requires
• The 30S ribosomal subunit
• The mRNA coding for the polypeptide to be made
• The initiating fMet-tRNA
• A set of three proteins called initiation factors (IF-1, IF-2, IF-3)
• GTP
• The 50S ribosomal subunit
• Mg ++
37
formation of initiation complex in prokaryotes

38. • Initiation occurs in three steps
1. step 1
• 30S ribosomal subunit binds two
initiation factors IF-1 and IF-3. .
• Factor IF-1 binds at the A site and
prevents tRNA binding at this site
during initiation.
• Factor IF-3 prevents the 30S and 50S
subunits from combining prematurely.
• The mRNA then binds to the 30S
subunit.
• The initiating (5’) AUG is guided to its
correct position by the Shine Dalgarno
Sequence in the mRNA. 38

39. • Shine Dalgarno Sequence is an initiation signal of 4 to 9 purine
residues. The sequence base-pairs with a complementary
pyrimidine-rich sequence near the 3’ end of the 16S rRNA of the
30S ribosomal subunit.
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40. • This mRNA-rRNA interaction positions the initiating (5’) AUG
sequence of the mRNA in the precise position on the 30S subunit
where it is required for initiation of translation.
• The particular (5’) AUG where fMet-tRNA is to be bound is
distinguished from other Methionine codons by its proximity to the
Shine Dalgarno sequence in the mRNA.
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• Bacterial ribosomes have three sites that bind tRNAs
-aminoacyl (A) site
-the peptidyl (P) site and
-exit (E) site
• the A and P sites bind to aminoacyl tRNAs whereas
• E site binds only to uncharged tRNAs that have completed their task on the
ribosome.

41. • The initiating (5’) AUG is positioned at the P site, the only site to
which fMet-tRNA can bind.
• The fMet-tRNA is the only aminoacyl-tRNA that binds first to the
P site; during the subsequent elongation stage, all other incoming
aminoacyl-tRNAs bind first to the A site and only subsequently to
the P and E sites.
• -E site is the site from which the “uncharged” tRNAs leave during
elongation
41

42. 2. step 2 - initiation process,
• the complex consisting of the 30S
ribosomal subunit, IF-3 and
mRNA is joined by both GTP-
bound IF-2 and the initiating
fMet-tRNA.
• The anticodon of this tRNA now
pairs correctly with the mRNA’s
initiation codon.
42

43. 3. step 3 - of initiation process,
• this large complex combines with the
50S ribosomal subunit; simultaneously,
the GTP bound to IF-2 is hydrolyzed to
GDP and Pi, which are released from the
complex.
• All these initiation factors depart from
the ribosome at this point.
• Completion of the steps in figure
produces a functional 70S ribosome
called the initiation complex, containing
the mRNA and the initiating fMet-tRNA.
43

44. • The correct binding of the fMet-tRNA to the P site in the
complete 70S initiation complex is assured by 3 points of
recognition and attachment that are;
1) The codon-anticodon interaction involving the initiation
AUG fixed in the P site
2) Interaction between Shine Dalgarno sequence in the
mRNA and the 16S rRNA
3) Binding interaction between the ribosomal P site and
fMet-tRNA
• The initiation complex is now ready for elongation.
44

45. 45
Formation of Initiation
complex
Fig: formation of initiation complex
in prokaryotes

46. Stage 3: Peptide bonds formation in elongation
stage
• The third stage of protein synthesis is elongation which
requires:
1. Initiation complex
2. Aminoacyl-tRNAs
3. A set of three soluble cytosolic proteins called the
elongation factors( EF-Tu, EF-Ts and EF-G) and
4. GTP
Cells use the three steps to add each amino acid residues, and the
steps are repeated as many times as there are residues to be
added.
46

47. Steps in elongation
• Elongation step 1: Binding of an incoming aminoacyl-
tRNA
• Elongation step 2: peptide bond formation
• Elongation step 3: Translocation
47

48. Elongation step 1:
Binding of an incoming aminoacyl-
tRNA
• appropriate incoming aminoacyL-
tRNA binds to a complex of GTP-
bound EF-Tu.
• Resulting aminoacyl - tRNA - EFTu-
GTP complex binds to the A site of
the 70S initiation complex.
• The GTP is hydrolyzed and EF-Tu-
GDP complex is released from the
70 S ribosome.
• The EF-Tu-GTP complex is
regenerated in a process involving
EF-Ts and GTP. 48

49. Elongation step 2: peptide
bond formation
• A peptide bond is now formed
between the two amino acids
bound by their tRNAs to the A
and P sites on the ribosomes.
• This occurs by the transfer of the
initiating N-formyl methionyl
group from its tRNA to the amino
group of the second aminoacyl-
tRNA in the A site, forming a
dipeptidyl-tRNA.
49

50. • At this stage, both tRNAs bound
to the ribosome shifts position in
the 50S subunit to take up a
hybrid binding state. The
uncharged tRNA shifts so that its
3’ and 5’ ends are in the E site.
Similarly, peptidyl-tRNA shift to
the P site.
• Peptidyl transferase that catalyzes
peptide bond formation is 23S
rRNA ribozyme.
50

51. Elongation step 3: Translocation
• the final step of elongation cycle,
the ribosome moves one codon
towards the 3’ end of the mRNA.
• Using energy provided by hydrolysis
of GTP bound to EF-G (translocase)
• This movement shifts the anticodon
of the dipeptidyl-tRNA, from the A
site to the P site, and shifts the
deacylated tRNA from the P site to
the E site, from where tRNA is
released into the cytosol.
51

52. • The third codon of mRNA now lies
in the A site and the second codon
in the P site.
• After translocation, the ribosome,
with its attached dipeptidyl-tRNA
and mRNA, is ready for the next
elongation cycle and attachment
of a third amino acid residue.
52

53. • Addition of next residue occurs in the same way as addition of
the second residue.
• The polypeptide remain attached to the tRNA of the most
recent amino acid to be inserted.
• The existing ester linkage between the polypeptide and tRNA is
broken during peptide bond formation, the linkage between
the polypeptide and the information in the mRNA persists,
because each newly added amino acid is still attached to its
tRNA.
53

54. Stage 4: Termination Of Polypeptide Synthesis
• Elongation continues until the ribosome adds the last amino
acid coded by the mRNA
• Termination, the fourth stage of polypeptide synthesis, is
signaled by the presence of one of three termination codons in
the mRNA (UAA, UAG, UGA) immediately following the final
coded amino acid.
54

55. • Once a termination codon occupies the ribosomal A site, three
termination factors or release factors- the proteins RF-1, RF-2 and
RF-3 contribute to:
• Hydrolysis of the terminal peptidyl tRNA bond
• Release of the free polypeptide and the last tRNA, now
uncharged, from the P site and
• Dissociation of 70 S ribosome into its 30S and 50S subunits,
ready to start a new cycle of polypeptide synthesis.
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• RF-1 recognizes the termination codons UAG and UAA.
• Rf-2 recognizes UGA and UAA.
• Either RF-1 or RF-2 binds at a termination codon and induces peptidyl
transferase to transfer the growing polypeptide to a water molecule rather
than to another amino acid.
• The specific function of RF-3 has not been firmly established.

56. • Hydrolysis of GTP by EF-G
leads to dissociation of the
50S subunit from the 30S-
tRNA –mRNA complex.
• The complex of IF-3 and
30S subunit is then ready
to initiate another round of
protein synthesis.
• EF-G and ribosome
recycling factor(RRF) are
replaced by IF-3 which
promotes the dissociation
of tRNA.
• The mRNA is then released.
56

57. STAGE 5: Post Translational Modifications
Folding And Processing
• It is a final stage of protein synthesis where nascent polypeptide
chain is folded and processed into its biologically active form.
• Some newly made proteins donot attain their final biologically
active conformation until they have been altered by one or more
processing reactions called post translational modifications.
57

58. 58

59. 59

60. 1. Amino-terminal and carboxyl-terminal modification:
• The first residue inserted in all polypeptides is N –
formylmethionine (in bacteria) or methionine (in eukaryotes).
• The formyl-group, the amino terminal Met residue and
additional amino terminal residues may be removed
enzymatically to form final functional protein.
2. Formation of disulfide cross-linkage:
• After folding into their native conformations, some proteins
form intrachain or interchain disulfide bridges between Cys
residues.
• This disulfide cross-linkage help to protect the protein
conformation from denaturation in the extracellular
environment.
60

61. 3. Loss of signal sequences:
• 15-30 residues at the amino terminal end of some proteins
called signal sequences which play a role in directing the protein
to its ultimate destination in the cell are removed by specific
peptidases.
4. Modification of individual amino acids:
• The hydroxyl groups of certain amino acids (Ser, Thr, and Tyr) are
enzymatically phosphorylated by ATP adding more negative
charge to polypeptide. The functional significance of this
modification varies from one protein to the next.
• For e.g: milk protein casein has many phosphoserine groups that
bind Ca2+
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62. 5. Attachment of carbohydrate side chains/ Glycosylation :
• The carbohydrate side chains of glycoproteins are attached
covalently during or after polypeptide synthesis.
• The complex carbohydrate moiety is attached to the amino acids,
serine and threonine (O- linked) or to asparagine (N-linked),
leading to the synthesis of gylcoproteins.
• Lubricating proteoglycans that coat mucous membrane, contains
oligosaccharide side chains.
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63. 6. Addition of isoprenyl groups
• The isoprenyl groups are derived from pyrophosphorylated
intermediates of the cholesterol biosynthestic pathway such as
farnesyl pyrophosphate.
• The isoprenyl group helps to anchor the protein in a membrane.
The carcinogenic activity of the ras oncogene is lost when
isoprenylation of the Ras protein is blocked.
• This has stimulated interest in identifying inhibitors of this
posttranslational modification pathway for use in cancer
chemotherapy.
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64. 7. Addition of prosthetic groups:
• Many proteins require covalently bound prosthetic groups.
• Two examples are the biotin molecule of acetyl-CoA carboxylase
and the heme- group of hemoglobin or cytochrome c.
8. Proteolytic procesing:
• Many proteins are initially synthesized as large, inactive precursor
of polypeptides that are proteolytically trimmed to form their
smaller, active forms.
• Examples include formation of insulin from preproinsulin , some
viral proteins, and proteases such as chymotrypsinogen and
trypsinogen.
64

65. Significance Of Translation Process
• Translation is the ultimate step of gene expression by which the
information of DNA carried by mRNA is decoded in the forms of
protein.
• It is also a detection tool in genetic recombinant technology by
which DNA of desired character is found to be present in cDNA
or not by expression selection.
• It is the tool by which proteins are synthesized from the amino
acids.
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66. 66

67. • 2077-03-19
• Utpal
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68. • Explain the process of translation (Prokaryotic
model) in detail. (using diagrams is preferable)
68