GENE EXPRESSION

INTRODUCTION
 GENE EXPRESSION
It is the process by which a gene's DNA
sequence is converted into the structures and
functions of a cell.
Non-protein coding genes are not translated
into protein.
Genetic information, chemically determined
by DNA structure is transferred to daughter cells
by DNA replication and expressed by
Transcription followed by Translation.
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• This series of events is called “Central
Dogma” is found in all cells and proceeds in
similar ways except in retroviruses which
posses an enzyme reverse transcriptase which
converts RNA into complementary DNA.
• Biological information flows from DNA to
RNA , and from there to proteins.
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THE CENTRAL DOGMA OF LIFE

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• Gene expression is a multi-step process which
involves
o Replication
o Transcription
o Translation

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 REPLICATION OF DNA
• It is a process in which DNA copies itself to
produce identical daughter molecules of DNA.
• DNA strands are antiparallel and complementary,
each strand can serve as a template for the
reproduction of the opposite strand.
• This process is called semiconservative replication.
• As the newly synthesized DNA has one half of the
parental DNA and one half of new DNA.

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• STEPS INVOLVED IN REPLICATION..
1. INITIATION.
2. ELONGATION.
3. TERMINATION

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 TERMINATION
• Termination occurs when DNA replication forks
meet one another or run to the end of a linear
DNA molecule.
• Also, termination may occur when a replication
fork is stopped by a replication terminator
protein.
• DNA Ligase fills up the gaps between the
Okazaki fragments.
• If mistake or damage occurs, enzymes such as a
nuclease will remove the incorrect DNA. DNA
polymerase will then fill in the gap . 9
 TRANSCRIPTION

• Transcription is the process through which a

DNA sequence is enzymatically copied by an
RNA polymerase to produce a complementary
RNA or in other words, the transfer of genetic
information from DNA into RNA.

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Transcription is divided into 3 stages.
• Initiation
• Elongation
• Termination
INITIATION
• RNA polymerase (RNAP) recognises and
binds to a specific region in the DNA called
promoter

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• There are two different base sequences on the
coding strand which the RNA polymerase
recognizes and for initiation:
• Pribnow box (TATA box) consisting of 6
nucleotide bases (TATAAT) and is located on
the left side about 10 bases upstream from the
starting point of the transcription.

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• The ‘-35’ sequence second recognition site in
the promoter region of the DNA and contains a
base sequence TTGACA which is located
about 35 bases upstream of the transcription
starting point.
• Closed complex RNAP binds to double
stranded DNA and this structure is called
Closed complex.
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 Open complex After binding of RNAP, the DNA

double helix is partially unwound and becomes single-

stranded in the vicinity of the initiation site. This

structure is called the open complex .

Elongation

 RNA synthesis then proceeds with addition of

ribonucleotide ATP, GTP, CTP and UTP as building
units.
 One DNA strand called the template strand serves as
the matrix for the RNA synthesis 15
• RNAP enzymes transcribe RNA in antiparallel direction
5’ → 3’. Transcription proceeds in complementary way :

 Guanine in DNA leads to Cytosine in RNA

 Cytosine in DNA leads to Guanine in RNA
 Thymidine in DNA leads to Adenine in RNA
 But Thymidine in DNA is replaced by Uracil in
RNA as consequence the
Adenine in DNA shows up for Uracil in RNA.

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• Different types of RNAPs

 RNA Polymerase I is located in the nucleolus and

transcribes ribosomal RNA (rRNA).

 RNA Polymerase II is localized to the nucleus, and

transcribes messenger RNA (mRNA) and most small
nuclear RNAs (snRNAs).

 RNA Polymerase III is localized to the nucleus (and

possibly the nucleolar- nucleoplasm interface), and
transcribes tRNA and other small RNAs 17
• Termination
• Two termination mechanisms are well known :-
 Intrinsic termination (Rho-independent
termination)
 Terminator sequences within the RNA that signal
the RNA polymerase to stop. The terminator
sequence is usually a palindromic sequence that
forms a stem-loop hairpin structure that leads to the
dissociation of the RNAP from the DNA template.
Example 'GCCGCCG'
 The RNA polymerase fails to proceed beyond this
point and the nascent DNA-RNA hybrid dissociates.
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 Rho-dependent termination uses a
termination factor called ρ factor (rho factor) to stop
RNA synthesis at specific sites.

 This protein binds and runs along the mRNA

towards the RNAP. When ρ-factor reaches the RNAP,
it causes RNAP to dissociate from the DNA and

terminates transcription.

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• Post transcriptional modification
• Post transcriptional modification is a process in
which precursor messenger RNA is converted into
mature messenger RNA (mRNA).
• The three main modifications are
I. 5' capping
II. 3' polyadenylation
III. RNA splicing

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 5' capping Addition of the 7 - Methylguanosine cap
to 5’ end is the first step in post-mRNA processing.
This step occurs co-transcriptionally after the
growing RNA strand has reached 30 nucleotides.
 3' polyadenylation The second step is the cleavage
of the 3' end of the primary transcript following by
addition of a polyadenosine (poly-A) tail.
 RNA splicing RNA splicing is the process by
which introns are removed from the mRNA and the
remaining exons connected to form a single
continuous molecule. The splicing reaction is
catalyzed by a large protein complex called the
spliceosome.
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 TRANSLATION
 It is a process by which proteins are synthesized.
Translation is a complex cellular process where
mRNA molecules, ribosomes, tRNA molecules,
amino acids, aminoacyl synthetases, energy
sources ATP and GTP and a number of factors
act together in a highly coordinated way.
 The mRNA carries genetic information
encoded as a ribonucleotide sequence from the
chromosomes to the ribosome.
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 The ribonucleotides are "read" by translational
machinery in a sequence of nucleotide triplets
called codons. Each of these triplet codes for a
specific amino acid. The ribosome and tRNA
molecules translate this code to produce proteins.
 tRNAs have a site for amino acid attachment,
and a site called an anticodon. These anticodon is
an RNA triplet complementary to the codons of
mRNA.
 Aminoacyl tRNA synthetase catalyzes the
bonding between specific tRNAs and the amino
acids that their anticodons sequences call for. The
product of this reaction is an aminoacyl-tRNA
molecule. 23
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• Initiation
Initiation of translation is divided into four
stages:-
• Dissociation of Ribosome
Initiation starts with the dissociation of the 80s
ribosome into 40s and 60s subunits.
Initiation factor IF-3 and IF-1A binds to the 40s
subunit and prevents its re-associaton with 60s
subunit.
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• Formation of 43s preinitiation complex
The first aminoacyl tRNA (fmet-tRNA)
binds to the 40s ribosomal subunit and forms
preinitiation complex. Initiation factor IF3 and
IF-1A stabilises this complex.
• Formation of 48s initiation complex
mRNA joins to the 43s preinitiation complex
and forms the 48s initaition complex. This step
requires energy from ATP.
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 Ribosomal initiation complex scans the
mRNA for the identification of the appropriate
initiation codon and its identification is
facilitated by specific sequence of nucleotide
surrounding it called Kozak Consensus
sequences.(ACCAUGG)
 In case of prokaryotes the recognition
sequence of initiation codon is referred to as
Shine-Dalgarno sequence.
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The Shine–Dalgarno (SD) sequence is a
ribosomal binding site in bacterial and The Kozak consensus sequence is a nucleic
archaeal messenger RNA, generally acid motif that functions as the protein
located around 8 bases upstream of the translation initiation site in most eukaryotic
start codon AUG. mRNA transcripts.

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• Formation of 80s initiation complex
 Initiation ends as the large 60s ribosomal
subunit joins the 48s initiation complex
causing the dissociation of initiation factors.
 The binding involves the hydrolysis of GTP.
 The step is facilitated by the involvement of
IF-5.

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• Elongation
• Elongation of the polypeptide chain involves addition of
amino acids to the carboxyl end of the growing chain.
During elongation the ribosome moves from the 5’ – end
to the 3’ – end of the mRNA that is being translated.
• Elongation is divided into Three steps:-
• Binding of aminoacyl-tRNA to A site
 The 80s initiation complex contains met-tRNA on the
P-site and the A-site is free.
 Another aminoacyl-tRNA recognises the codon on the
A-site and binds to it.
 This binding is facilitated by elongation factor-1α and
requires energy from GTP. 30
• Formation of peptide bond
 Now the P site contains the beginning of the
peptide chain of the protein to be encoded and the
A site has the next aminoacid to be added.
 The growing polypeptide connected to the tRNA
in the P site is detached from the tRNA in the P
site and a peptide bond is formed between the last
amino acids of the polypeptide and the amino
acid still attached to the tRNA in the A site.

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• Translocation
 Now, the A site has newly formed peptide,
while the P site has an unloaded tRNA (tRNA
with no amino acids).
 Then the ribosome moves 3 nucleotides
towards the 3' - end of mRNA.
 Since tRNAs are linked to mRNA by codon-
anticodon base-pairing, tRNAs move relative
to the ribosome taking the nascent polypeptide
from the A site to the P site and moving the
uncharged tRNA to the E exit site. This
process is catalyzed by elongation factor EF-2
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• Termination
 Termination occurs when one of the three
termination codons moves into the A site.
 These codons are recognized by proteins
called release factors, namely RF1
(recognizing the UAA and UAG stop codons)
or RF2 (recognizing the UAA and UGA stop
codons).

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• These factors trigger the hydrolysis of the ester
bond in peptidyl-tRNA and the release of the
newly synthesized protein from the ribosome.
At the same time the ribosome is dissociate
from the mRNA and recycled and used to
synthesise another protein.

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• Protein folding
 Protein folding is the process by which a
protein assumes its characteristic functional
shape or tertiary structure, also known as the
native state.
 All protein molecules are linear
heteropolymers composed of amino acids; this
sequence is known as the primary structure.

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 Most proteins can carry out their biological
functions only when folding has been
completed, because three-dimensional shape
of the proteins in the native state is critical to
their function.
 The process of folding often begins co-
translationally , so that the N-terminus of the
protein begins to fold while the C-terminal
portion of the protein is still being synthesized
by the ribosome.
 Specialized proteins called chaperones aid in
the folding of other proteins. 36
• Posttranslational modification
• Many proteins synthesized by translation are
not functional as such. Many changes takes
place in the protein after synthesis which
converts it into active protein. These are
known as post transcriptional modifications.

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• Trimming by Proteolytic Degradation
 Many proteins are synthesized as precursors
which are bigger in size than functional
proteins. Some portions of precursors is
removed by proteolysis to liberate active
protein . This process is called trimming.
 Example formation of insulin from
proinsulin.

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• Intein splicing
 Inteins are intervening sequences in proteins.
These are comparable to introns in mRNA.
Inteins have to be removed and exteins ligated
in the appropriate order for the protein to
become active.

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• Covalent Modifications
 Proteins synthesized by translation are
subjected to many covalent changes. By these
changes the proteins are converted to active or
inactive form. The covalent changes include
many modifications such as Phosphorylation,
hydroxylation, Glycosylation, Methylation,
Acetylation etc.

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 References

1.Biotechnology, by U. Sathyanarayana (page

number 38 – 58).
2.The molecular biology of cell by Albert,
Johnson,Lewis. 5th edition
3.Net source.

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