Genetic code

The genetic code is the set of rules by which information

encoded within genetic material (DNA or mRNA
sequence) is translated into proteins (amino acid
sequences) by living cells.
Biological decoding is accomplished by the ribosome
which links amino acids in an order specified by mRNA.
The code defines how sequences of these nucleotide
triplets, called codons, specify which amino acid will be
added next during protein synthesis.
The genetic code is highly similar among all organisms.
Discovery
Serious efforts to understand how proteins are encoded
began after the structure of DNA was discovered
by James Watson and Francis Crick.
George Gamow postulated that a three-letter code must
be employed to encode the 20 standard amino acids
used by living cells to build proteins.
With four different nucleotides, a code of 2 nucleotides
would allow for only a maximum of 42 or 16 amino acids.
A code of 3 nucleotides could code for a maximum of
43 or 64 amino acids.
Sequence Reading Frame
A codon is defined by the initial nucleotide from which
translation starts. For example, the string GGGAAACCC,
if read from the first position, contains the codons GGG,
AAA, and CCC; and, if read from the second position, it
contains the codons GGA and AAC; if read starting from
the third position, GAA and ACC.
Every sequence can, thus, be read in three reading
frames, each of which will produce a different amino acid
sequence.
Start/Stop Codons
Translation starts with a start codon. The most common
start codon is AUG, which is read as methionine or, in
bacteria, as formylmethionine. . Alternative start codons
(depending on the organism), include "GUG" or "UUG";
these codons normally represent valine and leucine,
respectively, but, as a start codon, they are translated as
methionine or formylmethionine.
The three stop codons are UAG, UGA and UAA. There is
no tRNA that has anticodon complementary to these
stop signals, and so protein synthesis terminates.
Start/Stop Codons
Mutation

During the process of DNA replication, errors

occasionally occur in the polymerization of the
second strand. These errors, called mutations,
can have an impact on the phenotype of an
organism.
Mutations may be point mutation or frame shift
mutation.
Point Mutation
A point mutation is a type of mutation that causes the
replacement of a single base nucleotide with another
nucleotide of the genetic material, DNA or RNA.
Point mutation may be of following type;
Nonsense mutation: A nonsense mutation converts
an amino acid codon into a termination codon. This
causes the protein to be shortened because of the stop
codon interrupting its normal code. How much of the
protein is lost determines whether or not the protein is
still functional.
Missense mutations: Code for a different amino acid.
A missense mutation changes a codon so that a different
protein is created.
Silent mutations: Code for the same amino acid. A
silent mutation has no effect on the functioning of
the protein A single nucleotide can change, but the new
codon specifies the same amino acid, resulting in an
unmutated protein.
Frame shift mutation

A type of mutation in which a number of nucleotides

deleted from or inserted into a protein coding sequence,
thereby causing an alteration in the reading frames of
the entire coding sequence downstream of the mutation.
The frame shift mutation will also alter the first stop
codon ("UAA", "UGA" or "UAG") encountered in the
sequence. The polypeptide being created could be
abnormally short or abnormally long, and will most likely
not be functional.
Characteristics
Universal: The genetic code is used by all species,
from prokaryotes to human being. However some
deviations are known to occur in mitochondria and some
unicellular organisms.
Directional: The sequence of triple-nucleotide is read
in the direction of 5’ to 3’. The first codon of coding
region of mRNA is almost always AUG, the initiation
codon. The last codon of the coding region of mRNA is
one of the stop codons.
Commaless: The coding region in mRNA is read in a
continuing way without punctuation. If there is insertion
or deletion of one or two nucleotides in the coding region
of mRNA frameshift mutation occurs.
Degeneracy: Because 61 codons specify 20 amino
acids, multiple codons must decode the same amino
acid. This is called degeneracy. For example Leu, Ser
and Arg each is specified by six different codons.
Non-overlapping: Code is non-overlapping. Each
nucleotide is part of only one codon and is read only
once.
DNA
replication
Topics covered:
Definition, Significance, Replication
Machinery, Process, Challenges
 Definition
DNA replication is the process by which a
double-stranded DNA molecule is copied to produce two
identical DNA molecules.

The process by which DNA makes a copy of itself during

cell division.

It is the process by which DNA is essentially doubled.

The Significance of DNA Replication

01 Ensures genetic continuity

02 Regulates reproduction of cells &

organisms

03 Required for regenertaion

04 Source of variation
 Replication Machinery

The cellular
machinery involved in
DNA replication is
comprised of 2 main
components
ENZYMES PROTEINS
 ENDONUCLEASE
An endonuclease produces an internal cut (single- or
double-stranded) in a DNA molecule. But a restriction
endonuclease produces cuts only at those sites that
have a specific base sequence.

During DNA replication, an endonuclease may induce a

nick to initiate DNA replication, or it may induce nicks to
generate a swivel (twist) for DNA unwinding.
 DNA GYRASE
(DNA TOPOISOMERASE)
DNA gyrase, or simply gyrase, is an enzyme within the
class of topoisomerase (Type II topoisomerase).

It relieves (removes) strain (stress/ pressure) while

double-stranded DNA is being unwound by helicase.
 DNA HELICASE
Helicase effects strand separation at the forks and use
one ATP molecule for each base that is separated.

In E. coli, DNA functions as helicase; this protein is a

hexamer and it moves with the replication fork.

Helicase breaks the hydrogen bonding between bases.

 SINGLE STRAND BINDING
PROTEINS (SSBP)
SSB protein binds to single-stranded DNA, and prevents
it from forming duplex DNA or secondary structures.

SSB binds as a monomer, but it facilitates the binding of

one SSB molecule facilitates binding of more SSB
monomers to the same DNA strand.

E. coli SSB is a tetramer.

 PRIMASE
This enzyme activity catalyzes the synthesis of RNA
primers to initiate DNA replication.

In E. coli, DnaG functions as primase.

But in eukaryotes, DNA polymerase α provides this

function. There are, however, several other ways in which
primers are produced, e.g., the 3′-OH generated by a nick
in the template DNA molecule.
 DNA POLYMERASE
(The Chief of All Replication Enzymes)
 DNA POLYMERASE
DNA polymerase is the chief enzyme of DNA replication.
DNA polymerase activity was discovered by
Arthur Kornberg in 1956.

E. coli has 4 more enzymes, DNA polymerase II, III, IV and

V;
DNA polymerase III (Pol III) is concerned with DNA
replication, while the remaining four enzymes are
involved in DNA repair.
 DNA POLYMERASE
All DNA polymerases require the following:

(1) A template DNA strand,

(2) A short primer (either RNA or DNA), and
(3) A free 3′ -OH in the primer.

They add one nucleotide at a time to the free 3′ -OH of

the primer, and extend the primer chain in 5′ → 3′
direction.
 DNA POLYMERASE
(Types)
The best‐studied bacterium, E. coli, has three DNA
polymerase types.

DNA polymerase I is simplest and best understood of

the DNA polymerases, not responsible for most
chromosomal DNA replication in E. coli,
but
functions in vivo both in DNA replication and DNA repair.
 DNA POLYMERASE
(Types)
DNA polymerase I has to perform 3 activities

1. The 5′ → 3′ polymerase activity is responsible for primer extension

or DNA synthesis.

2. The 5′ → 3′ exonuclease activity is involved in excision of DNA

strands during DNA repair; it removes ~ 10 bases at a time.
An exonuclease digests nucleic acids (here DNA) from one end, and
it does not cut DNA internally.

3. The 3′ → 5′ exonuclease activity is responsible for proof-reading.

 DNA POLYMERASE
(Types)
DNA polymerase II has to perform 2 activities

1. DNA polymerase II enzyme functions in DNA-repair.

2. It has 5′ → 3′ polymerase and 3′ → 5′ exonuclease activities, and

uses as template only such DNA duplexes that have short gaps.
 DNA POLYMERASE
(Types)
DNA polymerase III has to perform 2 activities

1. DNA polymerase III is involved in replication alongwith DNA

polymerase I.

2. DNA polymerase I and are single polypeptides but DNA polymerase III is a
10 subunits long protein with a molecular mass of 900KD).

3. It is responsible for DNA replication in vivo.

4. It has 5′ → 3′ polymerase and 3′ → 5′ exonuclease activities.

5. It catalyses polymeraization at very higher rates, 15,000 bases/ minute at

37⁰C.
 LIGASE
DNA ligase or polynucleotide ligase catalyzes the
formation of phosphodiester linkage between two
immediate neighbour nucleotides of a DNA strand.

Thus it seals the nicks remaining in a DNA strand either

following DNA replication or DNA repair.

However, this enzyme cannot fill the gaps in DNA

strands.
QUICK RECAP
 Terminology
Replication fork Origin of replication
Y-shaped area of highest A particular sequence in a
activity during replication. genome at which replication is
initiated.

Leading strand 5’ and 3’

Each strand of DNA or RNA has
That template strand at which a 5' end and a 3' end, named due
replication occurs in a continuous to the carbon position on
manner. the deoxyribose (or ribose) ring.

Lagging strand Klenow fragment

That template strand at which A large protein fragment produced
replication occurs in a dis-continuous after cleavage of DNA polymerase I
manner. by protease subtilisin.
Retains replication and repair
activities but in specific directions
 Terminology
Template / Parent strand RNA primer
Strand used by DNA polymerase to add A primer is a short single-stranded
new nucleotide bases complementary nucleotide stretch used by all living
to the existing / old DNA strand. organisms in the initiation of DNA
replication.

New / Daughter strand Upstream

Newly synthesized strand of DNA that is When considering double-stranded
copied via the addition of complementary
DNA, upstream is toward the 5' end
nucleotides from one strand of pre-existing
of the coding strand
DNA during DNA replication.

Okazaki fragments
Downstream
Short stretches of newly synthesized When considering
DNA found on the lagging strand double-stranded DNA,
during DNA replication. downstream is towards the 3' end.
Functional domains in the Klenow Fragment (left) and DNA
Polymerase I (PDB).
Steps of DNA
Replication
Process
 There are 4 main steps in replication process

1 Initiation 2 Primer Annealing 3 Elongation 4 Termination

Unwinding of Synthesis of primer at Addition of Daughter DNA strand
double helix DNA DNA template on the polynucleotides in is released while
template starnd site of origin of daughter strand by template strand
by helicase replication by primase DNA polymerase re-winds,
 DNA Replication in Prokaryotes
1. The two strands of DNA unwind at the origin of replication.
2. Helicase opens the DNA and replication forks are formed.
3. Helicase opens the DNA and replication forks are formed.

4. The DNA is coated by the single-strand binding proteins around the

replication fork to prevent rewinding of DNA.
5. Topoisomerase prevents the supercoiling of DNA.
6. RNA primers are synthesised by primase. These primers are complementary to
the DNA strand.

7. DNA polymerase III starts adding nucleotides at the end of the primers.
8. The leading and lagging strands continue to elongate.
9. The primers are removed and the gaps are filled with DNA Polymerase I and
sealed by ligase.
 Replisome
&
Replication fork

What is the difference?

The replisome is a large protein complex that carries out
DNA replication, starting at the replication origin.

It contains several enzymatic activities, such as helicase,

primase and DNA polymerase.

Replisome creates a replication fork to duplicate both the

leading and lagging strand.
Replication fork
Replication
Fork
 Challenges of Replication
Efficient replisome Many of the structural and chemical problems associated with DNA
replication are managed by molecular machinery that is highly
assembly is required ring
conserved across organisms. Ensu ssivity
se
proc

Separating duplex
lity
into leading & n in g h igh fide
i
lagging strands Mainta ut replication
g ho
throu
s+
proces

Protecting separated
strands from damage n through
Error correctio
se activity
3’-5’ exonuclea
of polymerase

Priming the leading & Synch

ronize
both s d poly
lagging strands Gap filling at trands meriza
Primer despit tion at
Okazaki nature e antip
removal arallel
fragments
 Useful animation links
DNA DNA
replication 3D replication
[Link]
[Link] hU-os8
WgcFPHqw
(Source: The Amoeba
(Source: McGraw Hill) Sisters)

Replication
Nucleotide
fork 3D
polymerization
[Link]
nDFeL7o [Link]
VyiEK0
(Source: McGraw Hill)
(Source: McGraw Hill)
 SUMMARY DNA Clamp is a
DNA duplication is protein that prevents
called replication elongating DNA
polymerase from
DNA polymerase cannot dissociating from
initiate replication, always growing strand

The assembly of depends on primer

molecular structures
(enzymes and proteins) Termination of
at origin of replication replication occurs
is called replisome when progress stops
at replication fork
Polymerases are highly
accurate : 1 mistake
/107 nucleotides added
Replisome creates Eukaryotes have
replication fork linear chromosomes

Replication occurs in 3
DNA polymerase is a Prokaryotes have
coordinated steps:
unidirectional enzyme circular chromosome
initiation, elongation
and termination
 References
BOOKS
1. Philip W. Kuchel, Ph.D; Simon Easterbrook-Smith; Vanessa Gysbers; J. Mitchell Guss. Schaum’s Outline of Biochemistry,
Third Edition. DNA Replication and Repair, Chapter (McGraw-Hill Companies, Inc., 2009, 1998, 1988).
[Link]
2. Victor W. Rodwell, David A. Bender, Kathleen M. Botham, Peter J. Kennelly, P. Anthony Weil. Harper's Illustrated
Biochemistry, 30edition, Chapter 35: DNA Organization, Replication, & Repair. (McGraw-Hill Companies, Inc., 2009)

WEB LINKS
I. [Link]
II. [Link]
III. [Link]
IV. [Link]
V. [Link]
VI. [Link]
VII. [Link]
VIII. [Link]
THANK YOU
Transcription
By Wajeeha Yaseen
Transcription

Cellular process in which RNA is

synthesized using DNA as a template known
as TRANSCRIPTION
Polymer of ribonucleotide held together by
3’ 5’ phosphodiester bridge
Is the only molecule known to function
both in the storage are single stranded. &
transmission of genetic information
Transcription
RNA
Three major kind of RNA
rRNA (60-80%). reads codes on mRNA and
transfers appropriate AA to mRNA.
tRNA (10-20%). transfer information of gene
to ribosome i.e. encodes the amino acids
sequence
mRNA (5-10%). Constituent of ribosome's
Many additional specialized RNAs which has
catalytic activity or regulatory functions are
present in the cell.
Transcription
In transcription a particular gene or group of
genes are copied at any time
In replication entire DNA molecule is normally
copied.
Gene is a segment of DNA that codes for a
type of Protein or for RNA some portions of
DNA are never transcribed. & may present on
any strand of DNA (contain many genes.)
Transcription
Feathers of transcription
1) It is highly selective.
This selectivity is due to signals embedded in
the nucleotide sequence of DNA.
Specific sequences mark the beginning and
end of the DNA segment which is to be
transcribed
This signals instruct the enzyme where to
start & stop the transcription when to start,
how often to start .
Transcription
2) Many of the RNA transcripts are
synthesized as precursors that is known as
primary transcripts
Which on modifications & trimming converted
into functional RNA
SITE
Prokaryotes– cytoplasm(all RNAs).
Eukaryotes– Nucleus & mitochondria
a) Nucleolus – rRNA
b) Nucleoplasm – tRNA & mRNA.
Transcription
RNA polymerase synthesize RNA in the
direction of 5’-3’ that means DNA template is
read in 3’-5’ direction
Ribonucleotides required -- ATP, GTP, CTP
The prokaryotic RNA polymerase is a
multimeric enzyme consisting of six subunits,
two identical α- subunits, similar but not
identical β and β’ and ω sixth is σ factor
2α,β,β’ω -- core enzyme
2α,β,β’ω + σ --- Holoenzyme
Transcription
RNA polymerase of prokaryotes
Subunit and function
α,α - Determine the DNA to be transcribed
β- Catalyze polymerization Bind
′β- bind and open the DNA template
ω- function is not known
Transcription
Function of RNAP
A single RNA polymerase performs multiple
functions in transcription process.
2- unwinds a short stretch of double helical DNA
3- selects correct [Link] to
promoter site & 5- interacts with activator
4- detects termination signals
catalyze the formation of phosphodiester bond
(polymerization according to base pair rule) &
repressor proteins that regulate the rate of
transcription.
Transcription
is the first step in gene expression. It involves
copying a gene's DNA sequence to make an
RNA molecule.
Transcription is performed by enzymes
called RNA polymerases, which link
nucleotides to form an RNA strand (using a
DNA strand as a template).
Transcription has three stages: initiation,
elongation and termination.
In eukaryotes, RNA molecules must be
processed after transcription
Transcription

The process of transcription can be broadly categorized

into 3 main stages:
initiation,
elongation &
termination.
Initiation

Transcription is catalysed by the enzyme RNA polymerase. It

attaches to and moves along the DNA molecule until it recognizes
a promoter sequence, which indicates the starting point of
transcription.
There may be multiple promoter sequences in a DNA molecule.
Transcription factors are proteins that control the rate of
transcription.
They too bind to the promoter sequences with RNA polymerase.
Once bound to the promotor sequence, RNA polymerase unwinds a
portion of the DNA double helix, exposing the bases on each of the
two DNA strands.
RNA polymerase builds an RNA strand in the 5' to 3'
direction, adding each new nucleotide to the 3' end of
the strand.
Elongation

One DNA strand (the template strand) is read in a 3′ to

5′ direction and so provides the template for the new
mRNA molecule.
The other DNA strand is referred to as the coding strand.
This is because the base sequence of the new mRNA is
identical to it, except for the replacement of thiamine
bases with uracil.
Incoming ribonucleotides are used by RNA polymerase to
form the mRNA strand. It does this using complementary
base pairing (A to U, T to A, C to G and G to C).
RNA polymerase then catalyses the formation of
phosphodiester bonds between adjacent ribonucleotides.
Bases can only be added to the 3′ (three-prime) end, so the
strand elongates in a 5’ to 3’ direction.
Termination

Elongation will continue until the RNA

polymerase encounters a stop sequence. At
this point, transcription stops and the RNA
polymerase releases the DNA template.
Sequences called terminators signal that the
RNA transcription is complete. Once they are
transcribed, they cause the transcript to be
released from the RNA polymerase.
video
[Link]
Translation
⚫ Protein synthesis is accomplished through a process
called translation.
⚫ After DNA is transcribed into a messenger RNA (mRNA)
molecule during transcription, the mRNA must be
translated to produce a protein.
⚫ In translation, mRNA along with transfer RNA(tRNA)
and ribosomes work together to produce proteins.
⚫ Stages of Translation in Protein Synthesis
⚫ Initiation: Ribosomal subunits bind to mRNA.
⚫ Elongation: The ribosome moves along the mRNA
molecule linking amino acids and forming a
polypeptide chain.
⚫ Termination: The ribosome reaches a stop codon,
which terminates protein synthesis and releases the
ribosome.
⚫ Transfer RNA
plays a huge role in protein synthesis and translation.
Its job is to translate the message within the
nucleotide sequence of mRNA to a specific amino acid
sequence.
⚫ These sequences are joined together to form a protein.
⚫ Transfer RNA is shaped like a clover leaf with three
loops.
⚫ It contains an amino acid attachment site on one end
and a special section in the middle loop called the
anticodon site.
⚫ The anticodon recognizes a specific area on a
mRNA called a codon.
⚫ Messenger RNA Modifications
⚫ Translation occurs in the cytoplasm.
⚫ After leaving the nucleus, mRNA must undergo several
modifications before being translated. Sections of the
mRNA that do not code for amino acids, called introns, are
removed.
⚫ A poly-A tail, consisting of several adenine bases, is added
to one end of the mRNA, while a guanosine triphosphate
cap is added to the other end.
⚫ These modifications remove unneeded sections and
protect the ends of the mRNA molecule.
⚫ Once all modifications are complete, mRNA is ready for
translation.
⚫ Translation
⚫ Once messenger RNA has been modified and is ready for
translation, it binds to a specific site on a ribosome.
⚫ Ribosomes consist of two parts, a large subunit and a
small subunit.
⚫ They contain a binding site for mRNA and two binding
sites for transfer RNA (tRNA) located in the large
ribosomal subunit.
Initiation
⚫ During translation, a small ribosomal subunit attaches
to a mRNA molecule. At the same time an initiator
tRNA molecule recognizes and binds to a
specific codon sequence on the same mRNA molecule.
A large ribosomal subunit then joins the newly formed
complex. The initiator tRNA resides in one binding
site of the ribosome called the P site, leaving the
second binding site, the A site, open. When a new
tRNA molecule recognizes the next codon sequence on
the mRNA, it attaches to the open A site. A peptide
bond forms connecting the amino acid of the tRNA in
the P site to the amino acid of the tRNA in
the A binding site.
 Elongation
⚫ As the ribosome moves along the mRNA molecule, the
tRNA in the P site is released and the tRNA in the A site
is translocated to the P site. The A binding site becomes
vacant again until another tRNA that recognizes the new
mRNA codon takes the open position.
⚫ This pattern continues as molecules of tRNA are
released from the complex, new tRNA molecules attach,
and the amino acid chain grows.
 Termination
⚫ The ribosome will translate the mRNA molecule until it
reaches a termination codon on the mRNA. When this
happens, the growing protein called a polypeptide chain is
released from the tRNA molecule and the ribosome splits
back into large and small subunits.
⚫ The newly formed polypeptide chain undergoes several
modifications before becoming a fully functioning protein.
⚫ Proteins have a variety of functions. Some will be used in
the cell membrane, while others will remain in the cytoplasm
or be transported out of the cell.
⚫ Many copies of a protein can be made from one mRNA
molecule. This is because several ribosomes can translate the
same mRNA molecule at the same time. These clusters of
ribosomes that translate a single mRNA sequence are called
polyribosomes or polysomes.
[Link]