Gene expression

1. 1. GENE EXPRESSION
2. 2. 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.
3. 3. • 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.
4. 4. THE CENTRAL DOGMA OF LIFE
5. • Gene expression is a multi-step process which involves o Replication o
Transcription o Translation
6. 6. 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.

7. 8. • STEPS INVOLVED IN REPLICATION.. 1. INITIATION. 2. ELONGATION. 3.

TERMINATION
8. 9. INITIATION  DNA replication starts at specific sites called Origin.  A specific
dna A protein binds with this site of origin and separates the double stranded DNA.
 Separation of two strands of DNA results in the formation of replication bubble
with a Replication Fork on either strands.  A Primer recognises specific
sequences of DNA in the replication bubble and binds to it.
9. 11. Helicase: The helicase unwinds the DNA helix by breaking the Hydrogen
bonds between the base pairs. Topoisomerase: The topoisomerases introduce
negative supercoils and relieve strains in the double helix at either end of the
bubble. The SSB proteins: The SSB proteins (Single Strands Binding) stabilize
the single strands thus preventing them to zip back together. 2/7/2016 11
10. 12. ELONGATION • DNA polymerase III binds to the Template strand at the 3’
end of the RNA Primer and starts polymerizing the nucleotides. • On leading
strand polymerization of nucleotides proceeds in 5’ – 3’ direction towards the
replication fork without interruption. • Lagging strand is replicated in 5’ – 3’
direction away from replication fork in pieces known as Okazaki Fragments. • As
DNA polymerase reaches the 5' end of the RNA primer of the next Okazaki
fragment; it dissociates and re-associates at the 3' end of the primer.2/7/2016 12
11. 13. • DNA polymerase I remove the RNA primers, and fills in with DNA. • DNA
ligase seals the nicks and connects the Okazaki fragments. • Helicase continues
 to unwind the DNA into two single strands ahead of the fork while topoisomerases
relieves the supercoiling caused by this.
12. 14. 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.
13. 15. 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.
14. 18. 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
15. 19. • There are two different base sequences on the coding strand which the RNA
polymerase recognises 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.
16. 20. • 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. 2/7/2016 20
17. 21.  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
2/7/2016 21
18. 22. • 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. 2/7/2016 22
19. 23. • 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
2/7/2016 23
20. 24. • 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. 2/7/2016 24
21. 25.  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. 2/7/2016 25
22. 26. • 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 2/7/2016 26
23. 27.  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. 2/7/2016 27
24. 28. 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. 2/7/2016 28
25. 29.  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.
26. 31. • 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.
27. 32. • 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.
28. 33.  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.  In case of
prokaryotes the recognition sequence of initiation codon is referred to as
Shine-Dalgarno sequence.
29. 34. • 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.
30. 35.
31. 36. • 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.
32. 37. • 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.

33. 39. • 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 2/7/2016 39
34. 40. • 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).
35. 41. • 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.
36. 42. • 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.
37. 43.  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.
38. 44. • 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.
39. 45. • 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.
40. 46. • 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.
41. 47. • 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.

42. 48. 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.
2/7/2016 48