The Genetic code, Translation and

Post-translational Modifications

Dr Mohamed Zahir
Department of Biochemistry,
Muhimbili University of Health and Allied Sciences, Tanzania
1
DNA 2

DNA stands for deoxyribose nucleic acid

This chemical substance is present in the nucleus

of all cells in all living organisms

DNA controls all the chemical changes which

take place in cells
DNA molecule 3

DNA is a very large molecule made up of a long

chain of nucleotides

Each nucleotide is made up of

• a sugar called deoxyribose

• a phosphate group and

• an organic base
The bases 5

The organic bases are

Adenine (A)

Thymine (T)

Cytosine (C)

Guanine (G)
Nucleotides 6

The deoxyribose, the phosphate and one of the bases

Combine to form a nucleotide

PO4

adenine

deoxyribose
Joined nucleotides 7

PO4

A molecule of DNA is
formed by millions of
nucleotides joined together in
PO4 a long chain

PO4

PO4

sugar-phosphate + bases
backbone
 8
DNA consists of a double strand of nucleotides

The sugar-phosphate chains are on the outside

and the strands are held together by bonds between the bases

PO4

PO4
2-stranded DNA 9
PO4
PO4

PO4
PO4

PO4 PO4

PO4
PO4

PO4
PO4

PO4
PO4

PO4
PO4

PO4
PO4
Bonding 1 10

The bases always pair up in the same way

Adenine forms a bond with Thymine

Adenine Thymine

and Cytosine bonds with Guanine

Cytosine Guanine
Bonding 2 11

PO4
PO4
adenine thymine

PO4
PO4
cytosine guanine

PO4
PO4

PO4
PO4
 13

The paired strands are coiled into a spiral called

A DOUBLE HELIX
 14

THE DOUBLE HELIX

bases

sugar-phosphate
chain
replication 16

Before a cell divides, the DNA strands unwind

and separate

Each strand makes a new partner by adding

the appropriate nucleotides

The result is that there are now two double-stranded DNA molecules in the
nucleus

So that when the cell divides, each nucleus

contains identical DNA

This process is called replication

 17
PO4
PO4 The strands
separate PO4
PO4

PO4 PO4

PO4 PO4

PO4
PO4

PO4
PO4

PO4
PO4

PO4
PO4
 18
Each strand builds up its partner by adding
the appropriate nucleotides
PO4 PO4
PO4 PO4

PO4 PO4
PO4
PO4

PO4 PO4
PO4 PO4

PO4 PO4 PO4

PO4

PO4 PO4
PO4 PO4

PO4 PO4
PO4
PO4

PO4 PO4
PO4 PO4

PO4 PO4
PO4
PO4
Genetic code 1 19

The sequence of bases in DNA forms the

Genetic Code

A group of three bases (a triplet) controls

the production of a particular amino acid in
the cytoplasm of the cell

The different amino acids and the order in which they are joined up
determines the sort of protein being produced
Genetic code 2 20

This is a small, imaginary protein molecule showing

how a sequence of 5 different amino acids could
determine the shape and identity of the molecule

Ser-Cyst-Val-Gly-Ser-Cyst Ala
Val
Val-Cyst-Ser-Ala-Ser-Cyst-Gly

Val- Cyst-Ala-Ala-Ser-Gly

Each amino acid (Serine, Cysteine, Valine, Glycine and

Alanine) is coded for by a particular triplet of bases
Coding 21
For example

Cytosine

Adenine Codes for Valine

Thymine

Cytosine (C)
Guanine (G) Codes for Alanine
Adenine (A)
Triplet code 22

This is known as the triplet code

Each triplet codes for a specific amino acid

CGA - CAA - CCA - CCA - GCT - GGG - GAG - CCA -

Ala Val Gly Gly Arg Pro Leu Gly

The amino acids are joined together in the correct

sequence to make part of a protein

Ala Val Gly Gly Arg Pro Leu Gly

DNA and enzymes 23

The proteins build the cell structures

They also make enzymes

The DNA controls which enzymes are made and

the enzymes determine what reactions take place

The structures and reactions in the cell determine

what sort of a cell it is and what its function is

So DNA exerts its control through the enzymes

Genes 24

A sequence of triplets in the DNA molecule may

code for a complete protein

Such a sequence forms a gene

There may be a thousand or more bases in

one gene
OVERVIEW OF TRANSLATION

• The second stage in gene expression is translating the nucleotide

sequence of a messenger RNA molecule into the amino acid
sequence of a protein.
• The genetic code is defined as the relationship between the
sequence of nucleotides in DNA (or its RNA transcripts) and the
sequence of amino acids in a protein.
• Each amino acid is specified by one or more nucleotide triplets
(codons) in the DNA.
• During translation, mRNA acts as a working copy of the gene in
which the codons for each amino acid in the protein have been
transcribed from DNA to mRNA.
➢tRNAs serve as adapter molecules that couple the
codons in mRNA with the amino acids they each specify,
thus aligning them in the appropriate sequence before
peptide bond formation.

➢Translation takes place on ribosomes, complexes of

protein and rRNA that serve as the molecular machines
coordinating the interactions between mRNA, tRNA, the
enzymes, and the protein factors required for protein
synthesis.

➢Many proteins undergo posttranslational modifications

as they prepare to assume their ultimate roles in the cell.
THE GENETIC CODE

Most genetic code tables designate the codons for amino

acids as mRNA sequences. Important features of the genetic
code include:
➢Each codon consists of three bases (triplet). There are 64
codons. They are all written in the 5' to 3' direction.
➢61 codons code for amino acids. The other three (UAA,
UGA, UAG) are stop codons (or nonsense codons) that
terminate translation.
➢There is one start codon (initiation codon), AUG, coding for
methionine. Protein synthesis begins with methionine (Met)
in eukaryotes, and formylmethionine (fmet) in prokaryotes.
➢The code is unambiguous. Each codon specifies no more
than one amino acid.
➢The code is degenerate. More than one codon can specify a single
amino acid.
➢All amino acids, except Met and tryptophan (Trp), have more than
one codon.
➢For those amino acids having more than one codon, the first two
bases in the codon are usually the same. The base in the third
position often varies.
➢The code is almost universal (the same in all organisms). Some
minor exceptions to this occur in mitochondria and some organisms.
➢The code is commaless(contiguous). There are no spacers or
"commas" between codons on an mRNA.
➢Neighboring codons on a message are non-overlapping.
Genetic Code
WoW Wobble Hypothesis

• Many amino acids are specified by more than one

codon (redundancy). Frequently, a tRNA can
translate more than one of these codons, sparing
the cell from making multiple tRNAs to carry the
same amino acid.
• For instance, the arg-tRNAarg can translate both
the CGA and the CGG codons that specify arginine.
This phenomenon is known as "Wobble" and can
be summarized as follows:
• Correct base pairing is required at the first position
of the codon (third of anticodon) and the second
position of the codon (second of anticodon).
• The third position of the codon does not always
need to be paired with the anticodon (e.g., it is
allowed to "wobble" in some cases).
Wobble and Protein Synthesis
MUTATIONS

• A mutation is any permanent, heritable change in the DNA

base sequence of an organism.
• This altered DNA sequence can be reflected by changes in
the base sequence of mRNA, and, sometimes, by changes in
the amino acid sequence of a protein.
• Mutations can cause genetic diseases. They can also cause
changes in enzyme activity, nutritional requirements,
antibiotic susceptibility, morphology, antigenicity, and
many other properties of cells.
• A transition is a point mutation that replaces a purine-
pyrimidine base pair with a different purine-
pyrimidine base pair. For example, an A-T base pair
becomes a G-C base pair.
• A transversion is a point mutation that replaces a
purine-pyrimidine base pair with a pyrimidine-purine
base pair. For example, an A-T base pair becomes a T-
A or a C-G base pair.
• Mutations are often classified according to the effect they
have on the structure of the gene's protein product.
• This change in protein structure can be predicted using the
genetic code table in conjunction with the base sequence of
DNA or mRNA.
Effect of Some Common Types of Mutations on Protein Structure
 Types of Mutations

Some Common Types of Mutations in DNA

 Overview of Eukaryotic Translation Initiation
Like transcription, translation is mechanistically divided into initiation, elongation, and
termination stages. All stages require translation factors in addition to ribosomes,
mRNA, and aa-tRNAs. Prior to initiation of translation, the 60S and 40S subunits of the
80S eukaryotic ribosome occur in their dissociated states. As described next, the
assembly of the 80S ribosome initiation complex at the start codon of the mRNA
proceeds via binding of the mRNA and a charged Met-tRNAiMet initiator tRNA to the 40S
subunit, with subsequent addition of the 60S subunit.
Translation Initiation in
Eukaryotes I
Translation initiation in eukaryotes
begins with three
components/complexes that are
shown near the top of Fig. 4.24. These
are 1) the 40S ribosomal subunit, to
which the eIF1, eIF1A, and eIF3
initiation factors are bound; 2) the
eIF2.GTP + Met-tRNAiMet ternary
complex; and 3) a circular mRNA
formed by the binding of the eIF4 cap-
binding complex at the 5’ end of the
mRNA to poly(A) binding protein
(PABP) associated with the 3’ end of
the mRNA. These components
associate in Steps 2 and 4 of the
diagram, placing Met-tRNAiMet in the P
site of the 40S subunit.
Translation Initiation in
Eukaryotes II
In the next stage of initiation, the
mRNA is scanned in the 5’ to 3’
direction until the first AUG start
codon is brought into the P site (Steps
5 & 6). Then the hydrolysis of GTP by
eIF2 generates a stable 48S initiation
complex in which the initiator tRNA
(Met-tRNAiMet) is H-bonded to the AUG
codon.
Translation Initiation in
Eukaryotes III
In the final stages of initiation, all
initiation factors except eIF1A
dissociate from the 48S initiation
complex and the 80S subunit and
eIF5B.GTP complex add on (Step 7).
After eIF5B hydrolyzes GTP, the last
initiation factors depart, and the stable
80S initiation complex is created (Step
8). This complex contains the complete
E (exit), P (peptidyl-tRNA), and A
(aminoacyl-tRNA) binding sites, with
Met-tRNAiMet bound to the P site.
Translation Elongation in
Eukaryotes
Translation elongation requires the assistance of
elongation factors (Fig. 4.25). In Step 1 of elongation,
the second amino acid of the polypeptide is carried
to the A site of the ribosome by an EF1a.GTP
complex. It binds to the mRNA via the anticodon
located in the A site. In Step 2, GTP is hydrolyzed and
EF1a departs. In Step 3, the 28S rRNA of the 60S
subunit catalyzes peptide bond formation (see Fig.
4.17), resulting in a dipeptidyl-tRNA residing in the A
site. In Step 4, the factor EF2.GTP binds, the
ribosome translocates one codon along the mRNA,
and GTP is hydrolyzed. As a result, the dipeptidyl-
tRNA is placed in the P site, and the uncharged
tRNAiMet enters the E site. The uncharged tRNA is
ejected from the ribosome in the next cycle of
elongation.
Translation Termination in
Eukaryotes
When a stop codon (UAA, UAG, UGA) enters the A
site, it is recognized and bound by the eRF1 release
factor (Fig. 4.27). eRF1 forms a complex with
eRF3.GTP. Hydrolysis of GTP by eRF3 results in
cleavage of the linkage between the polypeptide and
peptidyl-tRNA and release of the protein from the
ribosomal post-termination complex. A protein
called ABCE1 then binds to the complex, and via
ABCE1 hydrolysis of ATP, the 40S and 60S subunits
are separated. The 40S subunit recombines with the
eIF1, eIF1A, and eIF3 factors making it ready for
another round of initiation. Folding of the released
polypeptide chain is aided by chaperones (not
shown).
 Post Translational
Modifications of Proteins
Post-translational modifications are key mechanisms to increase
proteomic diversity. While the genome comprises 20,000 to 25,000 genes,
the proteome is estimated to encompass over 1 million proteins. Changes at
the transcriptional and mRNA levels increase the size of the transcriptome
relative to the genome, and the myriad of different post-translational
modifications exponentially increases the complexity of the proteome relative
to both the transcriptome and genome.
Types of Post Translational Modifications.
• Phosphorylation
• Glycosylation
• Ubiquitination
• S-Nitrosylation
• Methylation
• N-Acetylation
• Lipidation
• Proteolysis
 Phosphorylation
• 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.

Protein kinases
ATP + protein ———————> phosphoprotein + ADP
Phosphorylation
Example
 Glycosylation
• 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.
Example
 Classes of Glycans
• N-Linked glycans
• attached to nitrogen of Asparagine or arginine side chains.
• O-Linked glycans
• attached to hydroxy oxygen of serine,threonine
• Phospho glycans
• linked through the phosphate of serine.
• C-Linked glycans
• Rare form, Sugar is added to a carbon on tryptophan side chain.
 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.
Ubiquitin cycle
 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.
 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
 Example

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

 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
Example
Where,
HDACs = Histone deactyllase ,
KATs = N-acetyltransferase.
 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.
 Types of lipidation

• C-terminal glycosyl phosphatidylinositol (GPI) anchor

• N-terminal myristoylation
• S-palmitoylation
• S-prenylation
C-terminal glycosyl phosphatidylinositol (GPI)
anchor

• GPI anchors tether cell surface proteins to the plasma

membrane
• GPI-anchored proteins are often localized to
cholesterol- and sphingolipid-rich lipids, which act as
signaling platforms on the plasma membrane.
N-myristoylation

• It is the attachment of myristoyl group a 14-carbon

saturated fatty acid (C14) to a protein.
• It is facilitated by N-myristoyltransferase (NMT) and uses
myristoyl-CoA as the substrate.
S-palmitoylation

• It is addition of C16 palmitoyl group from palmitoyl-

CoA
• Palmitoyl acyl transferases (PATs) enzyme favors this
step.
• Reversed by thioesterases
S-prenylation

• Addition of a farnesyl (C15) or geranylgeranyl (C20)

group to proteins.
• Enzyme involved in this reaction is farnesyl
transferase (FT) or geranylgeranyl transferases (GGT I
and II).
Disulfide Bonding

• Disulfide bonds are covalent bonds formed between two

cysteine residues (R-S-S-R).
• These bonds contribute to the correct folding of proteins
as other elements of secondary structure
Disulfide Bonding
 Proteolysis

• Cleavage of peptide bonds by proteases.

• Examples of Proteases- Serine Proteases, Cysteine
Proteases, Aspartic acid Proteases.
• Involved in Antigen processing, Apoptosis, Cell
signalling
• Protein modifications can affect the protein involved
in several different ways:
• Change protein conformation to activate of inhibit
activity
• Alter association of the protein with other proteins in
the cell
• Induce relocation of the protein between
compartments of the cell
• All of the above at once.