The process of translation in a whole.

1. Protein Synthesis
Suvash C. Awal

2.  The last step in the gene expression
or the functional step of the genetic
information.
 The only step of central dogma
occurring exclusively in cytoplasm.

3. Requirement
 mRNA
 Aminoacyl-tRNA
 Ribosomes
 Others factors

4. Ribosomes
 Cell have tiny granular structures known as Ribosomes
 Ribosomes are Ribonucleo-Protein Particles
 Ribosomes serves as workbenches, with mRNA acting
as the blueprint in the process of protein synthesis.
 Ribosomes were first seen in cellular homogenates by
dark-field microscopy in the late 1930s by Albert Claude
who referred to them as “microsomes.” It was not until
the
mid-1950s, however, that George Palade observed
them in
cells by electron microscopy, thereby disposing of the
contention that they were merely artifacts of cell
disruption.
 In 1955, Paul Zamecnik demonstrate it as the site of

5.  The number of Ribosomes differs greatly
 A rapidly growing E.coli cell may have as many as
15,000 to 20,000 ribosomes, about 15% of the cell
mass
 Polyribosome (or polysome): an mRNA bearing
multiple ribosomes
 A single ribosome contacts with 30 nt, but mRNA can
bind one ribosome for every 80 nucleotides.

6. Structure
• Made up of RNA(rRNA) and
proteins
• 70S in prokaryotes and 80S in
eukaryotes
• Consist of two subunits
• Large subunit
• 50S in prokaryotes
• 60S in eukaryotes
• Small subunit
• 30S in prokaryotes
• 40S in eukaryotes

7. Comparison of bacterial and eukaryotic
ribosome

8. Secondary structure of rRNA

9. Svedberg Unit (S)
 The large and small subunit of ribosome are named according to
the velocity of sedimentation when subjected to centrifugal force.
 The unit used to measure sedimentation velocity is Sevdberg
(S).
 The larger value faster is the sedimentation velocity, hence larger
the molecule.
 Named after inventor of ultracentrifuge Theodor Sevdberg.
 The sedimentation velocity is a function of a particles molecular
weight, volume, size and shape

10. Functions
 Large subunit
◦ Peptidyl transferase center: that is
responsible for the formation of peptide
bonds
 Small subunit
◦ Decoding center: where charged tRNA
decode the codon units of the mRNA.
◦ Also the attachment of mRNA for the
initiation before large subunit join.

11. Prokaryotic mRNAs have a ribosome binding
site that recruits the translational machinery
 RBS (ribosome binding site): a short sequence upstream of the
start codon that facilitates binding of a ribosome. AGGAGG. Also
referred to as a Shine-Dalgarno sequence. Interacts with 16S
rRNA (3’ end containing anit Shine-Dalgarno sequence)
 In case that the start codon of downstream ORF overlaps the
stop codon of upstream ORF, for example, with AUGA,
translation of two ORFs is linked. This is known as translational
coupling.

12. Some translational initiation sequences recognized by E. coli ribosomes

13. Eukaryotic mRNAs are modified at their
5’ and 3’ ends to facilitate translation
 The 5’ cap is a methylated guanine nucleotide at 5’ end of
mRNA. Recruits the ribosome.
 The ribosome moves in a 5’ to 3’ direction until it encounters an
AUG in a process called scanning.
 The Kozak sequence (PuNNAUGG)(GCCRCCAUGG) interacts
with initiator tRNA. Poly-A tail promotes efficient recycling of
ribosomes
 Eukaryotic initiation factor and poly-A binding protein recognise
the 5’ cap and recruit the 43S rRNA forming preinitiaiton
complex.
 Cap-independent mode occur through the internal ribosome
entry site (IRES) recruiting ITAFs (IRES trans acting factor)

14. Polio virus IRES mediated initiation

15.  The large and Small subunits undergo
association and dissociation during each cycle
of Translation
 The ribosome cycle: the sequence of
association and dissociation of the ribosome.

16. Peptide bonds are formed by transfer of the
growing polypeptide chain from one tRNA to
another
 The ribosome catalyzes the formation of a peptide
bond between the amino acids attached to tRNAs.
 Two consequences of the peptidyl transferase
reaction: 1. The N-terminus of the protein is
synthesized before the C-terminus. 2.The growing
polypeptide chain is transferred from the peptidyl-
tRNA to the aminoacyl-tRNA.
 No ATP is required, but ATP is spent during tRNA
charging reaction.

18. Ribosomal RNAs are both structural and
catalytic determinants of the ribosome
 Ribosomal RNAs are not simply structural
components but directly responsible for catalytic
activity.
 The peptidyl transferase center and the decoding
center are composed almost entirely of RNA. Most
ribosomal proteins are on the periphery of the
ribosome.

19. The peptidyl transferase center

20. The decoding center

21. The ribosome has three binding
sites for tRNA
 The A site is for the aminoacylated-tRNA.
 The P site is for the peptidyl-tRNA.
 The E site is for the exiting tRNA.
 Each tRNA binding site is at the interface between
the large and the small subunits of the ribosome.

22. Channels through the ribosome allow the
mRNA and growing polypeptide to enter
and/or exit the ribosome
 There are two narrow channels in the small subunit,
one for entry and the other one for exit of mRNA. only
wide enough for unpaired RNA to pass through.
 There is a kink in the mRNA between the two codons.
The incoming aminoacyl tRNA cannot bind to bases
immediately adjacent to the vacant A site codon.
 A channel in the large subunit provides an exit path
for the newly synthesized polypeptide chain. The size
of the channel limits the folding of the growing
polypeptide chain. The polypeptide can form an alpha
helix in the channel.

24. In the ribosome there are THREE
STAGES and THREE operational SITES
involved in the protein production line.
 The three STAGES are
◦ Initiation,
◦ Elongation and
◦ Termination.
 The three operational or binding SITES are
◦ A site for attachment
◦ P site for peptide formation
◦ E site for exit (only on large subunit)

25. Table of binding sites, positions and functions in a
ribosome

26. Initiation

27. In prokaryotes
 The initiating 5’ AUG recruit tRNAfMet that code for
N-formylmethionine rather methionine in the
internal codon sequence by tRNAMet.
 The addition of N-formyl group to the amino group
of methionine by the transformylase prevent fMet
from entering interior positions in a polypeptide.

28. Requirement
 30S ribosomal subunit
 mRNA
 Initiating tRNAfMet
 Initiating factors (IF-1, IF-2, IF-3)
 GTP
 50S ribosomal subunit
 Mg++

29. It’s a three step process
 Step one
◦ 30S ribosomal subunit binds to IF-1 and IF-3(it
prevent combing 50S prematurely)
◦ 16S subunit recognize the Shine-Dalgarno
sequence and this mRNA-rRNA interaction drive
the positioning of intial 5’ AUG to the P site as A site
is blocked by IF-1.( the tRNAfMet only join to P site
and after that every other incoming aminoacyl-
tRNA join in A site)

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

32.  Step three
◦ 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 three initiation factors depart from the ribosome
at this point.
◦ Completion of the steps in produces a functional
70S ribosome called the initiation complex,
containing the mRNA and the initiating fMet-
tRNAfMet.

34. Translation initiation factors
 IF1 prevents tRNAs from binding to the portion of the small subunit
that will become part of the A-site.
 IF2 is a GTPase (a protein that binds and hydrolyzes GTP) that
interacts with three key components of the initiation machinery: the
small subunit, IF1, and the charged initiator tRNA (fMet-tRNAifMet).
By interacting with these components, IF2 facilitates the association
of fMet-tRNAifMet with the small subunit and prevents other
charged tRNAs from associating with the small subunit.
 IF3 binds to the small subunit and blocks it from reassociating with
a
large subunit. Because initiation requires a free small subunit, the
binding of IF3 is critical fora new cycle of translation. IF3 becomes
associated with the small subunit at the end of a previous round of
translation when it helps to dissociate the 70S ribosome into its
large and small subunits.

35. Initiation in Eukaryotes
 Eukaryotic initiation process is far more complicated
than prokaryotes, involving atleast 12 different
initiation factors (designated eIFn; “e” for eukaryotic)
 the small subunit is already associated with an
initiator tRNAwhen it is recruited to the capped 50
end of the mRNA. It then “scans” along the mRNA in
a 5’-3’ direction until it reaches the first 5’-AUG-3’
(kozak sequence).
 binding of the initiator tRNA to the small subunit
always precedes association with the mRNA

36. Initiation in Eukaryotes
43S preinitiation complex

41. Initiation factor required

43. Energy required
 For prokaryotes
◦ 1 ATP for aminoacyl-tRNA
◦ 1 GTP for initiation
 For eukaryotes
◦ 3 ATP, one for aminoacyl-tRNA, one for RNA
helicase, one for AUG scanning.
◦ 2 GTP, one in release of initiation factor by eIF2,
one by eLF5B for joining 60S larger subunit to 40S
small subunit forming complete 80S complex.

44. Elongatio
n

45. Elongation
 Ribosomes elongate polypeptide chains in a three-stage
reaction cycle that adds amino acid residues to a
growing polypeptide’s C-terminus
◦ Decoding, in which the ribosome selects and binds an
aminoacyl-tRNA, whose anticodon is complementary to the
mRNA codon in the A site.
◦ Transpeptidation, in which the peptidyl group on the P-site
tRNA is transferred to the aminoacyl group in the A site through
the formation of a peptide bond.
◦ Translocation, in which A-site and P-site tRNAs are respectively
transferred to the P site and E site accompanied by their bound
mRNA; that is, the mRNA, together with its base paired tRNAs, is
ratcheted through the ribosome by one codon.

48. Elongation factor
Eukaryotic counterpart
EF-Tu = eEF1A
EF-Ts = eEF1B
EF-G = eEF2

49. A Cycle of Peptide-Bond Formation Consumes
Two Molecules of GTP and One Molecule of ATP
 One molecule of nucleoside triphosphate (ATP) is
consumed by the aminoacyl-tRNA synthetase in
creating the high-energy acyl bond that links the amino
acid to the tRNA. The breakage of this high-energy
bond drives the peptidyl transferase reaction that
creates the peptide bond.
 A second molecule of nucleoside triphosphate (GTP) is
consumed in the delivery of a charged tRNA to the A-
site of the ribosome by EF-Tu and in ensuring that
correct codon–anticodon recognition had taken place.
 Finally, a third nucleoside triphosphate is consumed in
the EF-G-mediated process of translocation.

50. Termination

51.  Polypeptide formation end is signaled by the
presence of one of three termination codons
in mRNA (UAA, UAG, UGA).
 Once any of these codon take the A site the
termination process begins.
 tRNA doesn’t play role in it but the process
start with the involvement of termination
factors of release factors (RF)

52.  When a mutation produces a termination
codon in the interior of a gene, translation is
prematurely halted and the incomplete
polypeptide is usually inactive (nonsense
mutations).
 Gene can be restored to normal function if a
second mutation either
◦ Converts the misplaced termination codon to a
sense codon
◦ Suppresses the mutation by the mutation in tRNA
anticodon (nonsense supressors)

53. Release factor (RF)
 Stop codon recognition is depend upon the
RF.
 Two class of RF
◦ Class I RF = RF-1, RF-2
◦ Class II RF = RF-3
 Class I RF play role in recognition of stop
codon and act as catalyst for the hydrolysis
of peptidyl from peptidyl-tRNA
 Class II RF are small G-protein and act by
assisting class I RF in GTP-dependent
manner.

54. Difference from elongation
 In elongation codon recognition by aa-tRNA
result in nucleophillic reaction in the peptidyl
transferase centre (PTC), whereas in stop
codon recognition by RF leads to hydrolysis of
peptide-tRNA.
 Elongation process have proof-reading
mechanism to low frequency of error but RF in
termination is independent of proof-reading.
 Upon the stop-codon recognition the ester
bond of peptidyl-tRNA is cleaved by hydrolysis
in presence of water.

55.  In prokaryotes,
◦ RF-1 recognize UAA, UAG
◦ RF-2 recognize UAA, UGA
 The recognition depends upon the structure of RF-1,
RF-2.
 RF is made up of 4 Domain.
◦ Domain 1, bind to vicinity ribosomal GTPase associated
centre
◦ Domain II, role in stop codon recognition and also have
PxT and SPF that differentiate for RF-1 and RF-2
respectively.
◦ Domain III, spans between the functional centers of the
small and large ribosomal subunit; the universally
conserved GGQ motif implicated in the catalysis of peptidyl-
tRNA in the peptidyltransferase center.

56. Crystal structures of the 70S translation termination complexes bound with RF1 and

57.  The codon reading head of the release factor
comprises the N-terminal end of helix α5 and the
conserved recognition loop formed between the
β-4 and β-5 strands of the central β -sheet of
domain 2.
 Three elements of the reading head are
responsible for recognition of the three stop-
codon nucleotides.
◦ The N-terminal tip of helix α5 recognizes U via
formation of specific H-bonds from the backbone of
α5.
◦ Conserved amino acids of the recognition loop,
including the PxT and SPF motifs of RF1 and RF2,
respectively, define the specificity of release factors
for the second nucleotide (A & G).

58. Interactions of the first two stop-codon nucleotides with release factors RF1 and

59.  The N- and C-terminal ends of the
recognition loop define the specificity for the
third nucleotide located in the G530 pocket.
 Recognition of the third stop-codon
nucleotide by both RF1 and RF2 occurs
separately from the first two nucleotides, in
the G530 pocket of the decoding center.

60. Interactions of the third stop-codon nucleotide in the 70S translation terminatio
complexes bound with RF1 and RF2.

61. RF directly participate in
catalysis of peptidyl-tRNA
hydrolysis
 Upon recognition of a stop codon by RF, the
ester bond bridging peptidyl and peptidyl-
tRNA is hydorlyzed.
 Domain-III tip with GGQ motif inserted into
the PTC and contact the nucleotide of 23s
rRNA, P-site, tRNA and it open a passage
for a water molecule.

63. Process
 Recognition of stop codon by RF-1/RF-2.
 Peptidyl hydrolysis form peptidyl-tRNA with
introduction of water molecule.
 Once the newly synthesized polypeptide has been
released from the ribosome, the class II release factor
RF-3, in its complex with GDP, binds to the ribosome.
On binding to the ribosome–RF-1/2 complex, it
exchanges its bound GDP for GTP. The resulting
change in the conformation of RF-3, as seen in cryo-
EM studies, causes it to bind more tightly to the
ribosome and expel the RF-1/2

64.  The interaction of RF-3⋅GTP with the ribosome
stimulates it to hydrolyze its bound GTP. The
resulting RF-3⋅GDP then dissociates from the
ribosome. Subsequently, ribosomal recycling factor
(RRF) binds in the ribosomal A site followed by EF-
G⋅GTP.
 EF-G hydrolyzes its bound GTP, which causes RRF
to be translocated to the P site and the tRNAs
previously in the P and E sites to be released.
Finally, the small and large ribosomal subunits
separate, a process that is facilitated by the binding
of IF-3, and RRF, EF-G⋅GDP, and mRNA are
released. The ribosomal subunits can then
participate in a new round of initiation

66. Stop-codon recognition and peptidyl-tRNA
hydrolysis are coordinated via
conformational switch in release factor
 Class I release factors are high-fidelity enzymes.
 In order to achieve low error frequency (10-3-10-6),
hydrolysis of peptidyl-tRNA has to be strictly
coordinated with stop-codon recognition.
 This coordination is accomplished by preventing
the docking of domain 3 into the PTC prior to
recognition of a stop codon.
 a release factor initially interacts with the ribosome
in a catalytically inactive conformation. Upon stop-
codon recognition, a conformational change would
occur resulting in the docking of the GGQ motif into
the PTC

68. GTP Hydrolysis Speeds Up
Ribosomal Processes
 What is the role of the GTP hydrolysis reactions
mediated by the various ribosomally associated G
proteins (IF-2, EF-Tu, EF-G, and RF-3 in
bacteria)?
 The high rate and irreversibility of the GTP
hydrolysis reaction ensures that the various
complex ribosomal processes to which it is
coupled, initiation, elongation, and termination, will
themselves be fast and irreversible.
 The ribosome utilizes the free energy of GTP
hydrolysis to gain a more ordered (lower entropy)
state rather than a higher energy state as often
occurs in ATP-dependent processes

69. Reference
 Voet, D. & Voet, J. G. “ Biochemistry”. Fourth Edition.
2011. John Wiley & Sons. INC.
 Watson, et. al. “Molecular Biology of The Gene”.
Seventh Edition. 2014. Pearson Education, Inc.
 Korostelev, A. a. (2011). Structural aspects of
translation termination on the ribosome. RNA (New
York, N.Y.), 17(8), 1409–1421.
 Jackson, R. J., Hellen, C. U. T., & Pestova, T. V.
(2010). The mechanism of eukaryotic translation
initiation and principles of its regulation. Nature
Reviews. Molecular Cell Biology, 11(2), 113–127.