Here I had describing translation and post translational modification in a simple way for under graduates and post graduates

2. Translation,
1. mRNA : RBS, start-stop codon ,
2. Ribosome : rRNA + protein + protein
3. tRNA : tRNA charging charging
4. Amino acid : genetic code

3. • Translation, the second part of the central
dogma of molecular biology, describes how
the genetic code is used to make amino acid
chains.
• In this lesson, explore the mechanics involved
in polypeptide synthesis.
• Learn the three major steps of translation as
you watch tRNA, mRNA, and ribosomes go to
work.

4. • Translation is the process that takes the information
passed from DNA as messenger RNA and turns it into a
series of amino acids bound together with peptide
bonds.
• It is essentially a translation from one code (nucleotide
sequence) to another code (amino acid sequence).
• The ribosome is the site of this action, just as RNA
polymerase was the site of mRNA synthesis.
• The ribosome matches the base sequence on the
mRNA in sets of three bases (called codons) to tRNA
molecules that have the three complementary bases in
their anticodon regions.

5. • Again, the base-pairing rule is important in this
recognition (A binds to U and C binds to G). The
ribosome moves along the mRNA, matching 3
base pairs at a time and adding the amino acids
to the polypeptide chain.
• When the ribosome reaches one of the "stop"
codes, the ribosome releases both the
polypeptide and the mRNA. This polypeptide will
twist into its native conformation and begin to act
as a protein in the cells metabolism.

7. • To understand basic steps in translation are:
• The ribosome binds to mRNA at a specific area.
• The ribosome starts matching tRNA anticodon
sequences to the mRNA codon sequence.
• Each time a new tRNA comes into the ribosome, the
amino acid that it was carrying gets added to the
elongating polypeptide chain.
• The ribosome continues until it hits a stop sequence,
then it releases the polypeptide and the mRNA.
• The polypeptide forms into its native shape and starts
acting as a functional protein in the cell.

8. Types of RNA:
• mRNA - messenger RNA is a copy of a gene. It acts as a photocopy
of a gene by having a sequence complementary to one strand of
the DNA and identical to the other strand. The mRNA acts as a
busboy to carry the information stored in the DNA in the nucleus to
the cytoplasm where the ribosomes can make it into protein.
• tRNA - transfer RNA is a small RNA that has a very specific
secondary and tertiary structure such that it can bind an amino acid
at one end, and mRNA at the other end. It acts as an adaptor to
carry the amino acid elements of a protein to the appropriate place
as coded for by the mRNA.
• rRNA - ribosomal RNA is one of the structural components of the
ribosome. Its sequence is the compliment of regions in the mRNA
so that the ribosome can match with and bind to an mRNA it will
make a protein from.

11. The Three Steps of Translation
• Translation is the second step in the central
dogma that describes how the genetic code is
converted into amino acids.
• We've talked about how the mRNA codes are
recognized by tRNA and how the amino acids are
linked together by peptide bonds.
• A chain of amino acids is also called a
polypeptide. Polypeptides are assembled inside
the ribosomes, which are tiny organelles on the
rough ER of a cell.

12. • Now that we're learning more about the
mechanics of translation, we're going to have
to start putting the pieces together.
• We already understand the role of the
ribosome and the amino acids in the process
of translation, but how does polypeptide
assembly actually occur? .

13. There are three important steps to the
process of translation
• There's a
• Beginning step, initiation,
• a middle step, called elongation,
• a final step, called termination.

14. • These three words may sound familiar to you. The
same terms are used in transcription to describe the
steps involved in making the mRNA strand.
• But, here in translation, we're making a polypeptide
strand.
• In either case, we're making a long molecule out of a
chain of smaller subunits.
• So, whether we're referring to transcription or
translation, the three terms accurately describe the
mechanics of the process.
• Let's walk through each step, one at a time

15. Initiation
• We'll start with initiation. During initiation, the mRNA,
the tRNA, and the first amino acid all come together
within the ribosome.
• The mRNA strand remains continuous, but the true
initiation point is the start codon, AUG. Remember that
the start codon is the set of three nucleotides that
begins the coded sequence of a gene.
• Remember also that the start codon specifies the
amino acid methionine. So, methionine is the name of
the amino acid that is brought into the ribosome first.

17. And, how did methionine get itself to the ribosome? By attaching
to the tRNA that contains the right anticodon.
The anticodon for AUG is UAC.
We know that because of the rules of complementary base
pairing.
The tRNA with the anticodon UAC will automatically match to the
codon AUG, bringing the methionine along for the ride.
So, there you have it - mRNA is attached to tRNA, and tRNA is
attached to methionine. That's initiation.

19. Elongation
• The next step makes up the bulk of
translation. It's called elongation, and it's the
addition of amino acids by the formation of
peptide bonds.
• Elongation is just what it sounds like: a chain
of amino acids grows longer and longer as
more amino acids are added on. This will
eventually create the polypeptide.

20. • Now that we've begun with the start codon, the mRNA
shifts a little through the ribosome so that the next
codon is up for grabs.
• Let's say the next codon is UAU. So, now we need a
tRNA that has the matching anticodon, AUA. Oh, look!
Here's a tRNA with the right anticodon, and it's
brought along a tyrosine.
• Tyrosine is the amino acid that is specified by the
codon UAU.
• The tRNA attaches to the mRNA in the ribosome and
lines up tyrosine right next to the waiting methionine.
A peptide bond forms between the two amino acids.

21. • Then, the first tRNA leaves everyone else behind
and floats off to find more work to do. Poor
methionine! Now it's just drifting around like a
lonely kite in the wind! That tRNA left methionine
hanging by only one anchor: its peptide bond
with tyrosine.
• The tyrosine is still attached to its own tRNA,
which, in turn, is clinging to the mRNA inside the
ribosome.
• Already we can see the beginnings of a
polypeptide elongating outward.

22. • Should we walk through that process one more time?
Let's keep everything just as we have it here and move
on to add our third amino acid. mRNA shifts over again,
and now the third codon is ready for a match.
• What's that codon? CAC. Here comes a tRNA with the
matching anticodon, GUG.
• It's also brought us a histidine, since CAC codes for
histidine.
• The tRNA's anticodon matches up with the mRNA's
codon, putting the histidine in perfect position for
making a peptide bond with tyrosine.

23. The ribosome then moves one triplet forward
and a new tRNA+amino acid can enter the
ribosome and the procedure is repeated.

24. How is the right tRNA Chosen
• Non-matching tRNA's may also enter the A site.
• Why doesn't this cause wrong amino acids to be added to
the chain?
• Well, non-matching tRNA's may be able to enter the A
site...but in general, they don't get to stay there. In a
careful process that never fails to fascinate me, each tRNA
is escorted by helper proteins, and only a tRNA that's a
perfect match for the codon will be "released" into the A
slot by its choosy helpers.^7​7​​start superscript, 7, end
superscript
• A molecule of the energy storage molecule guanosine
triphosphate (GTP) is used up to release the tRNA, which is
why the diagram shows this step as needing GT

25. What are the N and C terminus
• Great question! Amino acids have an amino
group {−NH​2} at one end and of a carboxyl
group {-COOH} at the other:

26. • A polypeptide is made up of amino acids
connected in a chain.
• Although the amino and carboxyl groups of
most amino acids in the chain will be tied up
in peptide bonds, the amino acids at the very
ends of the chain will each have a free
chemical group.

27. • One end of the polypeptide has an exposed
amino group. This end is called the N-
terminus ,for the nitrogen atom of the amino
group.
• The other end of the polypeptide has an
exposed carboxyl group. This end is called
the C-terminus, for the carbon atom of the
carboxyl group.

29. • The first amino acid in a polypeptide (the
methionine carried by the first tRNA) lies at
the N-terminus, and new amino acids are
progressively added at the C-terminus:

30. • But...odds are we may want a longer
polypeptide than two amino acids. How does
the chain continue to grow? Once the peptide
bond is formed, the mRNA is pulled onward
through the ribosome by exactly one codon.
This shift allows the first, empty tRNA to drift
out via the E ("exit") site. It also exposes a new
codon in the A site, so the whole cycle can
repeat.

31. Termination
• Translation ends when one of three stop codons,
UAA, UAG, or UGA, enters the A site of the
ribosome.
• There are no aminoacyl tRNA molecules that
recognize these sequences.
• Instead, release factors bind to the P site,
catalyzing the release of the completed
polypeptide chain and separating the ribosome
into its original small and large subunits

32. • To meet this requirement they make many
mRNA copies of the corresponding gene and
have many ribosomes working on each mRNA.
After translation the protein will usually
undergo some further modifications before it
becomes fully active.

33. At a given time, more than one ribosome may
be translating a single mRNA molecule. The
resulting clusters of ribosomes, which resemble
beads on a string, are called polysomes

34. • When the ribosome reaches one of three stop codons, for
example UGA, there are no corresponding tRNAs to that
sequence.
• Instead termination proteins bind to the ribosome and
stimulate the release of the polypeptide chain (the protein),
and the ribosome dissociates from the mRNA.
• When the ribosome is released from the mRNA, its large and
small subunit dissociate. The small subunit can now be loaded
with a new tRNA+methionine and start translation once again.
• Some cells need large quantities of a particular protein.

35. Easy To Remember Translation Steps
Step 1 of Translation : mRNA attaches to the ribosome.
Step 2 of Translation : tRNA's attach to free amino acids in the cytoplasmic
"pool" of amino acids.
step 3of Translation t: RNA carries its specific amino acid to the ribosome.
Step 4 of Translation : tRNA "delivers" its amino acid based on
complementary pairing of a triplet code (anticodon) with the triplet code
(codon) of the mRNA.
Step 5 of Translation : Enzyme "hooks" the amino acid to the last one in the
chain forming a peptide bond.
Step 6 of Translation : Protein chain continues to grow as each tRNA brings
in its amino acid and adds it to the chain.

36. Elongation & Termination of protein biosynthesis

37. 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.
•

38. • 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.)

39. • 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

40. 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.

41. 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 amino acyl 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

42.  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.

43. 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.

44. • Once the protein has been synthesized by the
ribosome from its corresponding mRNA in the
cytosol, many proteins get directed towards the
endoplasmic reticulum for further modification.
• Certain N and C terminal sequences are often
cleaved in the ER after which they are modified
by various enzymes at specific amino acid
residues. These modified proteins then undergo
proper folding to give the functional protein.

45. • Most of the proteins that are translated from mRNA
undergo chemical modifications before becoming
functional in different body cells. The modifications
collectively, are known as post-translational
modifications.
• The protein post translational modifications play a
crucial role in generating the heterogeneity in proteins
and also help in utilizing identical proteins for different
cellular functions in different cell types.
• How a particular protein sequence will act in most of
the eukaryotic organisms is regulated by these post
translational modifications.

46. • Post translational modifications occurring at the
peptide terminus of the amino acid chain play an
important role in translocating them across
biological membranes.
• These include secretory proteins in prokaryotes
and eukaryotes and also proteins that are
intended to be incorporated in various cellular
and organelle membranes such as
• lysosomes,
• chloroplast,
• mitochondria and plasma membranes.

47. • Expression of proteins is important in diseased
conditions. Post translational modifications play
an important part in modifying the end product
of expression and contribute towards biological
processes and diseased conditions.
• The amino terminal sequences are removed by
proteolytic cleavage when the proteins cross the
membranes.
• These amino terminal sequences target the
proteins for transporting them to their actual
point of action in the cell.

48. Why PTM is necessary
• Stability of protein.
• Biological activity.
• Protein targeting.
• Protein signaling.

49. • As noted above, the large number of different
PTMs precludes a thorough review of all possible
protein modifications.
• Therefore, this overview only touches on a small
number of the most common types of PTMs
studied in protein research today.
• Furthermore, greater focus is placed on
phosphorylation, glycosylation and
ubiquitination, and therefore these PTMs are
described in greater detail on pages dedicated to
the respective PTM

50. Importance of PTMs
• Play a crucial role in generating the
heterogeneity in proteins.
• Help in utilizing identical proteins for different
cellular functions in different cell types.
• Regulation of particular protein sequence
behavior in most of the eukaryotic organisms.

51. • Play an important part in modifying the end
product of expression.
• Contribute towards biological processes and
diseased conditions.
• Translocation of proteins across biological
membranes

52. • There are several types of post translational
modifications that can take place at different amino
acid residues.
• The most commonly observed PTMs include
phosphorylation,
• glycosylation,
• methylation as well as
• hydroxylation and acylation.
• Many of these modifications, particularly
phosphorylation, serve as regulatory mechanisms for
protein action.

53. Phosphorylation
• Reversible protein phosphorylation, principally on
serine, threonine or tyrosine residues, is one of the
most important and well-studied post-translational
modifications.
• Phosphorylation plays critical roles in the regulation of
many cellular processes including cell cycle, growth,
apoptosis and signal transduction pathways.
• Phosphorylation is the most common mechanism of
regulating protein function and transmitting signals
throughout the cell. While phosphorylation has been
observed in bacterial proteins, it is considerably more
pervasive in eukaryotic cells.

54. • It is estimated that one-third of the proteins in
the human proteome are substrates for
phosphorylation at some point (1). Indeed,
phosphoproteomics has been established as a
branch of proteomics that focuses solely on
the identification and characterization of
phosphorylated proteins.

55. Addition of phosphate group to a
protein.
Principally on serine, threonine or
tyrosine residues.
Also known as Phospho regulation.
Critical role in cell cycle, growth,
apoptosis and signal transduction
pathways.

56. • Phosphorylation of amino acid residues is carried out
by a class of enzymes known as kinases that most
commonly modify side chains of amino acids
containing a hydroxyl group.
• Phosphorylation requires the presence of a phosphate
donor molecule such as ATP, GTP or other
phoshorylated substrates.
• Serine is the most commonly phosphorylated residue
followed by threonine and tyrosine.
• Removal of phosphate groups is carried out by the
phosphatase enzyme and thus this forms one of the
most important mechanisms for regulation of proteins.

57. Glycosylation
• Glycosylation involves the enzymatic addition of
saccharide molecules to amino acid side chains. This
can be of two types – N-linked glycosylation, which
links sugar residues to the amide group of aspargine
and Olinked glycosylation, which links the sugar
moieties to the hydroxyl groups of serine or threonine.
• Suitable glycosyl transferase enzymes catalyze these
reactions.
• Sugar residues that are attached most commonly
include galactose, mannose, glucose,
Nacetylglucosamine, Nacetylgalactosamie as well as
fucose

58. The covalent attachment of oligosaccharides
Addition of glycosyl group or carbohydrate group to a
protein.
Principally on Asparagine, hydroxylysine, serine or
threonine.
Significant effect on protein folding, conformation,
distribution, stability and activity

59. Hydroxylation
• During the formation of collagen, the
amino acids proline & lysine are
respectively converted to hydroxyproline
& hydroxylysine.
• This hydroxylation occurs in the
endoplasmic reticulum & requires
vitaminC.

60. Ubiquitination
• Ubiquitin is a small regulatory protein that can
be attached to the proteins and label them for
destruction.
• Effects in cell cycle regulation, control of
proliferation and differentiation, programmed
cell death (apoptosis), DNA repair, immune
and inflammatory processes and organelle
biogenesis.

61. Ubiquitin cycle

62. S-Nitrosylation
• Nitrosyl (NO) group is added to the protein.
• NO a chemical messanger that reacts with free
cysteine residues to form S-nitrothiols.
• Used by cells to stabilize proteins, regulate
gene expression.

64. Alkylation/Methylation
• Addition of methyl group to a protein.
• Usually at lysine or arginine residues.
• Binds on nitrogen and oxygen of proteins
• Methyl donor is S-adenosylmethionine (SAM)
• Enzyme for this is methyltransferase
• Methylation of lysine residues in histones in
DNA is important regulator of chromatin
structure

65. SAM (S-adenosyl methionine) is converted into SAH(S-adenosyl homocysteine)

66. N-Acetylation
• Addition of acetyl group to the nitrogen.
• Histones are acetylated on lysine residues in
the N-terminal tail as a part of gene
regulation.
• Involved in regulation of transcription factors,
effector proteins, molecular chaperons and
cytoskeletal proteins.
• Methionine aminopeptidase (MAP) is an
enzyme responsible for N-terminal acetylation

68. Lipidation
• Lipidation attachment of a lipid group, such as
a fatty acid, covalently to a protein.
• In general, lipidation helps in cellular
localization and targeting signals, membrane
tethering and as mediator of protein-protein
interactions

69. Identification of PTM modifications
• Mass spectrometry
• HPLC analysis
• Incorporation of radioactive groups by addition to
growing cells
– e.g., 75Se-labeling and chromatographic
isolation of proteins
• Antibody cross-reactivity
– e.g., antibody against phosphotyrosine
• Polyacrylamide gel electrophoresis (PAGE