Gene expression

1. GENE EXPRESSION
2/7/2016 1

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.
2/7/2016 2

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.
2/7/2016 3

4. THE CENTRAL DOGMA OF LIFE
2/7/2016 4

5. • Gene expression is a multi-step process which
involves
o Replication
o Transcription
o Translation
2/7/2016 5

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.
2/7/2016 6

7. 2/7/2016 7

8. • STEPS INVOLVED IN REPLICATION..
1. INITIATION.
2. ELONGATION.
3. TERMINATION
2/7/2016 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.
2/7/2016 9

10. 2/7/2016 10

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

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

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.
2/7/2016 13

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.
2/7/2016 14

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.
2/7/2016 15

16. 2/7/2016 16

17. 2/7/2016 17

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
2/7/2016 18

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.
2/7/2016 19

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

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

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

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

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

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

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

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

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

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.
2/7/2016 29

30. 2/7/2016 30

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.
2/7/2016 31

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.
2/7/2016 32

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.
2/7/2016 33

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.
2/7/2016 34

35. 2/7/2016 35

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.
2/7/2016 36

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.
2/7/2016 37

38. 2/7/2016 38

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

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).
2/7/2016 40

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.
2/7/2016 41

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.
2/7/2016 42

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.2/7/2016 43

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.
2/7/2016 44

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.
2/7/2016 45

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.
2/7/2016 46

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.
2/7/2016 47

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

49. 2/7/2016 49