The present ppt is covers all aspects of protein translation in bacteria as well as in eukaryotes. It also includes a brief introduction to ribosomes and tRNA which are among the key components of the translation machinery.

1. PROTEIN
Translation
West Bengal State University
cbcs Botany Core viii
Dr. Riddhi Datta
Department of Botany
Dr. A.P.J. Abdul Kalam Government
College

2. Translation
• The information for the proteins found in a cell is encoded in genes of the
genome of the cell.
• A protein-coding gene is expressed by transcription of the gene to produce an
mRNA, followed by translation of the mRNA.
• Translation involves the conversion of the base sequence of the mRNA
into the amino acid sequence of a polypeptide.
• The base sequence information that specifies the amino acid sequence of a
polypeptide is called the genetic code.
Dr. Riddhi Datta

3. Translation
• Polypeptide synthesis takes place on
ribosomes, where the genetic message
encoded in mRNA is translated.
• The mRNA is translated in the 5’-to-3’
direction.
• The polypeptide is made in the N-
terminal–to–C-terminal direction.
• Amino acids are brought to the ribosome
bound to tRNA molecules.
Dr. Riddhi Datta

4. • Polypeptide synthesis takes place on ribosomes.
• Several thousands of ribosomes are present in each cell.
• Ribosomes bind to mRNA and facilitate the binding of the tRNA to the
mRNA (codon-anticodon pairing) so that a polypeptide chain can be
synthesized.
• In both prokaryotes and eukaryotes, ribosomes consist of two unequally sized
subunits—the large and small ribosomal subunits.
Dr. Riddhi Datta
RIBOSOME
• Each subunit contains one or more
rRNA molecules and a large
number of ribosomal proteins.

5. Bacterial ribosome:
• Size of 70S (S = sedimentation coefficient/ Sevedberg unit)
• Consists of two subunits of sizes 50S (large subunit) and 30S (small subunit)
• Contain three rRNA molecules—
– 23S rRNA and 5S rRNA in the large subunit
– 16S rRNA in the small subunit
Dr. Riddhi Datta
RIBOSOME

6. Eukaryotic (mammalian) ribosome:
• Size of 80S
• Consists of two subunits of sizes 60S (large subunit) and 40S (small subunit)
• Contain four rRNA molecules—
– 28S rRNA, 5.8S rRNA, and 5S rRNA in the large subunit
– 18S rRNA in the small subunit
Dr. Riddhi Datta
RIBOSOME

7. • During translation, the mRNA passes through
the small subunit of the ribosome.
• It has 3 binding sites for tRNAs:
– A (aminoacyl) site is where an incoming
aminoacyl–tRNA binds and consists of
both small and large subunits
– P (peptidyl) site is where the tRNA
carrying the growing polypeptide chain is
located and consists of both small and
large subunits
– E (exit) site is where a tRNA binds on its
path from the P site to leaving the
ribosome and consists only of large
subunit
Dr. Riddhi Datta
RIBOSOME

8. • The regions of DNA that
contain genes for rRNA are
called ribosomal DNA (rDNA)
or rRNA transcription
units.
• E. coli has 7 rRNA
transcription units.
• Each rRNA transcription unit
contains single promoter
and one copy each of the
16S, 23S, and 5S rRNA
coding sequences, arranged
in the order 16S–23S–5S.
Dr. Riddhi Datta
RIBOSOMal RNA genes IN BACTERIA

9. • Transcription by RNA polymerase
produces a precursor rRNA (pre-
rRNA) molecule which also contain
some non-rRNA spacer
sequences.
• The pre-rRNA is processed to
remove the spacers, releasing the
three rRNAs.
• The transcript-processing events
take place in a large
ribonucleoprotein complex and
specific associations of the rRNAs
with ribosomal proteins generate
the functional ribosomal subunits.
Dr. Riddhi Datta
RIBOSOMal RNA genes IN BACTERIA

10. • The genes for 18S, 5.8S, and 28S
rRNAs are found adjacent to one
another in the order 18S–5.8S–28S,
with each set tandemly repeated
100 to 1,000 times to form clusters
of rDNA repeat units.
• Due to active transcription of the
repeat units, a nucleolus forms
around each cluster and the multiple
nucleoli then fuse to form one
nucleolus.
• Each eukaryotic rDNA repeat unit is
transcribed by RNA polymerase I to
produce a pre-rRNA molecule which
has some non-rRNA spacer
sequences.
Dr. Riddhi Datta
RIBOSOMal RNA genes IN EUKARYOTES

11. • The 5S rRNA is produced by transcription of
the 5S rRNA genes (typically located
elsewhere in the genome) by RNA
polymerase III.
• The pre-rRNA is processed to remove the
spacers, releasing the three rRNAs.
• The pre-rRNA processing takes place in
complexes formed between the pre-rRNA,
5S rRNA, and ribosomal proteins which
ultimately forms the functional 60S and 40S
ribosomal subunits and are then transported
to the cytosol.
Dr. Riddhi Datta
RIBOSOMal RNA genes
IN EUKARYOTES

12. Transfer RNA
• During translation of mRNA, each
transfer RNA (tRNA) brings a specific
amino acid to the ribosome which is to be
added to a growing polypeptide chain.
• The correct amino acid sequence of a
polypeptide is achieved as a result of:
– the binding of each amino acid to
a specific tRNA
– the binding between the codon of
the mRNA and the complementary
anticodon in the tRNA.
Dr. Riddhi Datta

13. Structure of Transfer RNA
• tRNAs are 75 to 90 nucleotides long having a
cloverleaf structure.
• The differences in tRNA sequences explain the ability
of a particular tRNA molecule to bind a specific
amino acid.
• The cloverleaf results from complementary base
pairing between different sections of the molecule,
producing 4 base-paired ―stems‖ separated by 4
loops: I, II, III, and IV.
• Loop II contains the three-nucleotide anticodon
sequence, which pairs with a three-nucleotide codon
sequence in mRNA during translation.
Dr. Riddhi Datta

14. Structure of Transfer RNA
• All tRNAs exhibit an upside-down L-shaped 3D
structure in which the 3’ end of the tRNA is at
the end of the L that is opposite from the
anticodon loop.
• All tRNA molecules have the sequence 5’-CCA-3’
at their 3’ ends where the amino acid
attaches.
• All tRNA molecules have a number of
enzymatically modified bases, which include
– I: inosine,
– T: ribothymidine,
– ψ: pseudouridine,
– D: dihydrouridine,
– Gme: methylguanosine,
– GMe2: dimethylguanosine,
– Ime: methylinosine. Dr. Riddhi Datta

15. Transfer RNA GENES
• tRNA genes in bacteria:
– found in one or a few copies in the genome
– transcribed by the only RNA polymerase found in bacteria
• tRNA genes in eukaryotes:
– repeated many times in the genome
– transcribed by RNA polymerase III
• Transcription of tRNA genes in both bacteria and eukaryotes produces precursor
tRNA (pre-tRNA) molecules, which are then post-transcriptionally modified, a 5’-
CCA-3’ sequence is added to the 3’ end and modification of bases throughout the
molecule then take place.
• Some tRNA genes in eukaryotes may contain introns between the first and second
nucleotides 3’ to the anticodon.
Dr. Riddhi Datta

16. Aminoacylation of trna
• The peptide bond formation between the amino group and carboxyl group of
two amino acids is thermodynamically unfavorable. This barrier is overcome
by the activation of carboxyl group of the precursor amino acid.
• The correct amino acid is attached to the tRNA by an enzyme called
aminoacyl–tRNA synthetase.
• The process is called aminoacylation, or charging or activation, and
produces an aminoacyl–tRNA (or charged tRNA).
• Aminoacylation uses energy from ATP hydrolysis.
• There are 20 different aminoacyl–tRNA synthetases, one for each of the 20
different amino acids.
• Each enzyme recognizes particular structural features of the tRNA or tRNAs it
aminoacylates.
Dr. Riddhi Datta

17. Aminoacylation of trna
1
2
3
4
5
Dr. Riddhi Datta

18. Aminoacylation of trna
1. First, the amino acid and ATP bind to the specific aminoacyl–tRNA synthetase enzyme.
2. The enzyme then catalyzes a reaction in which the ATP is hydrolyzed to AMP, which covalently
binds to the amino acid (carboxyl group) as AMP to form aminoacyl–AMP.
L-aa (L-amino acid) + ATP + ENZ (Enzyme) aa-AMP-ENZ (aminoacyl-AMP-enzyme complex) + PPi
3. Next, the tRNA molecule binds to the enzyme.
4. The enzyme then transfers the amino acid from the aminoacyl–AMP to the tRNA, displaces the
AMP and releases the aminoacyl–tRNA molecule. This is called tRNA charging.
tRNA-3’OH + aa-AMP-ENZ tRNA-aa (aminoacyl-tRNA) + AMP + ENZ
5. The enzyme then returns to its original state.
 Chemically, the amino acid attaches at the 3’ end of the tRNA by a covalent linkage between
the carboxyl group of the amino acid and the 3’-OH or 2’-OH group of the ribose of the
adenine nucleotide found at the 3’ end of every tRNA.
https://vimeo.com/138715154
Dr. Riddhi Datta

19. PROTEIN SYNTHESIS
• Requirements of protein synthesis:
– A pool of 20 amino acids
– 20 aminoacyl-tRNA synthetases
– ATP and GTP
– Different tRNA molecules
– Ribosomes
– Initiation factors
– Transfer factors
– Elongation factors
– Release factors
– Mg2+ ions
– Several co-factors
• The three basic stages of protein synthesis are—
– Initiation
– Elongation
– Termination
• Apart from these, aminoacylation of tRNA is required to initiate translation.
Dr. Riddhi Datta

20. Initiation of Translation
• Initiation encompasses all of the steps preceding the formation of the peptide
bond between the first two amino acids in the polypeptide chain.
• Initiation involves:
– an mRNA molecule
– a ribosome
– a specific initiator tRNA
– protein initiation factors (IF)
– GTP
– Mg2+ ions
Dr. Riddhi Datta

21. Initiation of Translation IN BACTERIA
• Initiation of translation begins with the
formation of the initiation complex.
• The complex consists of:
– 30S ribosomal subunit
– three initiation factors, IF1, IF2 and IF3
– mRNA molecule to be translated
– GTP
– Mg2+ ions
• First, the 30S subunit interacts with IF1
and IF3 and then binds with the mRNA
molecule near the translation start site.
• IF3 aids in the binding of the 30S subunit to
mRNA and prevents binding of the 50S
ribosomal subunit to the 30S subunit.
Dr. Riddhi Datta

22. Initiation of Translation IN BACTERIA
• The AUG initiating codon signals the start of translation where the 30S subunit actually
binds.
• Binding also requires a sequence upstream of the AUG, called the ribosome binding site
(RBS) or Shine-Dalgarno sequence.
• It is a purine rich sequence (AGGAG or similar) and is complementary to a pyrimidine rich
region (UCCUC) at the 3’ end of the 16S rRNA in ribosome.
• Most RBS are 8-12 nucleotides upstream from AUG.
• There is a formation of complementary base pairs between the mRNA and 16S rRNA of the 30S
subunit which locates the RBS of the mRNA for the initiation of protein synthesis.
Dr. Riddhi Datta

23. Initiation of Translation IN BACTERIA
• The next step is the binding of a special initiator tRNA to the AUG start codon to which
the 30S subunit is bound.
• In both prokaryotes and eukaryotes, the AUG initiator codon specifies methionine.
• In many cases, the methionine is removed later.
• In bacteria, the initiator tRNA is tRNA.fMet, which has the anticodon 5’-CAU-3’ to bind
to the AUG start codon.
• This tRNA carries a modified form of methionine, formylmethionine (fMet).
• First, methionyl–tRNA synthetase catalyzes the addition of methionine to the tRNA. Then
the enzyme transformylase adds the formyl group to the amino group of methionine.
• The resulting molecule is designated fMet–tRNA.fMet.
• When an AUG codon in an mRNA molecule is encountered at a position other than the
start of the amino acid-coding sequence, a different tRNA, called tRNA.Met, is used to
insert methionine at that point in the polypeptide chain.Dr. Riddhi Datta

24. Initiation of Translation IN BACTERIA
• The initiator tRNA, fMet–tRNA.fMet, is
brought to the 30S subunit–mRNA complex
by IF2, which also carries a molecule of GTP.
• The 70S ribosome has 3 binding sites for
aminoacyl-tRNA:
– Exit (E) site
– Peptidyl (P) site
– Aminoacyl (A) site
• The initiator tRNA binds to the subunit in the P
site guided by IF2.
• IF1 blocks the A site.
• IF3 prevents association of the 50S subunit.
• This forms the 30S initiation complex.
Dr. Riddhi Datta

25. Initiation of Translation IN BACTERIA
• Next, the 50S ribosomal subunit binds, leading
to GTP hydrolysis and the release of the three
initiation factors.
• This final complex is called the 70S initiation
complex.
Dr. Riddhi Datta
https://www.youtube.com/watch?v=3ooX
hZqboqE

26. Initiation of Translation IN Eukaryotes
• Very similar to that in bacteria
• However the process is more complex
• It involves many more initiation factors, called eukaryotic initiation factors (eIF)
• Differences:
– The initiator methionine is unmodified, although a special initiator tRNA
brings it to the ribosome.
– Shine–Dalgarno sequences are not found in eukaryotic mRNAs. Instead, the
initiation complex forms at the 5’ end of the mRNA.
Dr. Riddhi Datta

27. Initiation of Translation IN Eukaryotes
• A cap-binding protein (CBP) binds to the 7-
methyl guanosine cap at the 5’ terminus of the
mRNA.
• Then, other initiation factors (eIFs) bind to the
CBP-mRNA complex.
• Then the small (40S) subunit of the ribosome
binds.
• Eukaryotes contain a special initiator tRNA,
tRNAiMet (―i‖ for initiator), but the amino group
of the methionyl-tRNAiMet is not formylated.
• The initiator methionyl-tRNAiMet interacts
with a soluble eIF and enters the P site directly
during the initiation process, just as in E. coli.
Dr. Riddhi Datta

28. Initiation of Translation IN Eukaryotes
• The entire initiation complex moves 5’ → 3’ along the mRNA molecule, searching
for an AUG codon.
• The AUG codon is embedded in a short sequence—called the Kozak sequence, after
Marilyn Kozak—which indicates that it is the initiator codon.
• The optimal initiation sequence is 5’-GCC(A or G)CCAUGG-3’.
• This process is called the scanning model for initiation.
• When an AUG triplet is found, the initiation factors dissociate from the complex.
• Now the large (60S) subunit binds to the methionyl-tRNA/mRNA/40S subunit
complex, forming the complete (80S) ribosome.
• The 80S ribosome/mRNA/tRNA complex is ready to begin the second phase of
translation, chain elongation.
Dr. Riddhi Datta

29. Elongation of Translation
• The elongation involves addition of amino acids to the growing
polypeptide chain one by one.
• This phase has three steps:
– Aminoacyl–tRNA (charged tRNA) binds to the ribosome in the A
site.
– A peptide bond forms.
– The ribosome moves (translocates) along the mRNA one codon.
• Elongation requires accessory protein factors, called elongation factors
(EF), and GTP.
• Elongation is similar in eukaryotes.
Dr. Riddhi Datta

30. Elongation of Translation
STEP 1:
Binding of Aminoacyl–tRNA
• The anticodon of fMet–tRNA is hydrogen bonded to the AUG initiation
codon in the P site of the ribosome.
• The next codon in the mRNA is in the A site (Ex: UCC codon specifies
serine) where the appropriate aminoacyl–tRNA binds (here, Ser–
tRNA.Ser).
• This aminoacyl–tRNA is brought to the ribosome bound to EF-Tu–GTP
complex.
• When the aminoacyl-tRNA binds to the codon in the A site, GTP
hydrolysis releases EF-Tu–GDP and EF-Tu is recycled.
Dr. Riddhi Datta

31. Elongation of Translation
STEP 1:
Recycling of EF-Tu:
• First, a second elongation factor, EF-Ts, binds to EF-Tu and displaces
the GDP.
• Next, GTP binds to the EF-Tu–EF-Ts complex to produce an EF-Tu–GTP
complex simultaneously releasing EF-Ts.
• Another aminoacyl-tRNA binds to the EF-Tu–GTP, and the entire process
is repeated.
• The process is highly similar in eukaryotes, with eEF-1A playing the role
of EF-Tu, and eEF-1B playing the role of EF-Ts.
Dr. Riddhi Datta

32. Elongation of Translation
Dr. Riddhi Datta
STEP 1:

33. Elongation of Translation
STEP 2:
Peptide Bond Formation:
• The ribosome maintains the two aminoacyl–
tRNAs in the P and A sites in the correct
positions for a peptide bond formation.
• First, the bond between the amino acid and the
tRNA in the P site (here, fMet and its tRNA) is
cleaved.
• Second, the peptide bond is formed between the
now-freed fMet and the Ser attached to the
tRNA in the A site in a reaction catalyzed by
peptidyl transferase.
• The 23S rRNA molecule of the large ribosomal
subunit functions as the peptidyl transferase
activity.
Dr. Riddhi Datta
STEP 2:

34. Elongation of Translation
STEP 2:
Peptide Bond Formation:
• Once the peptide bond has formed, a tRNA without an attached amino acid (an uncharged
tRNA) is left in the P site.
• The tRNA in the A site, now called peptidyl–tRNA, has the first two amino acids of the
polypeptide chain attached to it (here, fMet–Ser).
Dr. Riddhi Datta

35. Elongation of Translation
STEP 3:
Translocation:
• In the last step in the elongation cycle, the ribosome moves one codon along
the mRNA toward the 3’ end.
• Translocation requires the activity of another elongation factor, EF-G.
• The steps involve:
– EF-G–GTP complex binds to the ribosome
– GTP is hydrolyzed
– translocation of ribosome occurs displacing the uncharged tRNA
away from the P site
• Translocation is similar in eukaryotes; the elongation factor in this case is
eEF-2, which functions like bacterial EF-G.
Dr. Riddhi Datta

36. Elongation of Translation
STEP 3:
Translocation:
• During the translocation step, the peptidyl–tRNA remains attached to its
codon on the mRNA.
• As the ribosome moves, the uncharged tRNA moves from the P site to the E
site.
• The peptidyl–tRNA is now located in the P site.
• When translocation is complete, the uncharged tRNA is then released from
the ribosome and EF-G is released and recycled.
• The A site is also vacant where an aminoacyl–tRNA with correct anticodon
binds to the newly exposed codon.
Dr. Riddhi Datta

37. Dr. Riddhi Datta
Elongation of Translation
STEP 3:

38. Elongation of TranslationSTEP 3:
Translocation:
• The entire elongation process is then repeated until a stop codon is encountered.
• In both bacteria and eukaryotes, once the ribosome moves away from the
initiation site on the mRNA, another initiation event occurs on another ribosome.
Dr. Riddhi Datta
• Typically, several
ribosomes are
translating each
mRNA
simultaneously
together forming a
complex called
polysome or
polyribosome.
https://www.youtube.com/watch?v=bNFKt6RVPf4

39. TERMINATION of Translation
• The termination of translation is signaled by one of
three stop codons: UAG, UAA, and UGA.
• Stop codons are same in prokaryotes and
eukaryotes.
• The stop codons do not code for any amino acid, so
have no tRNAs for them.
• The ribosome recognizes a stop codon with the help
of proteins called release factors (RF).
• In E. coli, there are three RFs: RF1, RF2 and RF3.
• RF1 recognizes UAA and UAG and RF2
recognizes UAA and UGA.
Dr. Riddhi Datta

40. TERMINATION of Translation
• The binding of RF1 or RF2 to a stop
codon triggers peptidyl transferase to
cleave the polypeptide from the tRNA
in the P site.
• The polypeptide then leaves the
ribosome.
Dr. Riddhi Datta

41. TERMINATION of Translation
• Next, RF3–GDP binds to the
ribosome, stimulating the release of
the RF from the stop codon and the
ribosome.
• GTP now replaces the GDP on RF3.
• RF3 hydrolyses the GTP which
allows RF3 to be released from the
ribosome.
Dr. Riddhi Datta

42. TERMINATION of
Translation
• In E. coli, ribosome recycling factor
(RRF) binds to the A site.
• Then EF-G binds, causing translocation
of the ribosome and thereby moving
RRF to the P site and the uncharged
tRNA to the E site.
• The RRF releases the uncharged tRNA,
and EF-G releases RRF, causing the two
ribosomal subunits to dissociate from the
mRNA.
Dr. Riddhi Datta

43. TERMINATION of Translation
• In eukaryotes, the termination process is similar to that in bacteria.
• In this case, a single release factor— eukaryotic release factor 1 (eRF1)—
recognizes all three stop codons, and eRF3 stimulates the termination
events.
• Ribosome recycling occurs in eukaryotes, but there is no equivalent of RRF.
Dr. Riddhi Datta
https://www.youtube.com/watch?v=mjAuhjJMCws