2. What is molecular biology?
Is the study of biology at a molecular level.
The field overlaps with other areas of biology, particularly genetics and
biochemistry
Molecular biology concerns itself with understanding the interactions
between the various systems of a cell, including the interrelationship of DNA,
RNA and protein synthesis and learning how these interactions are regulated.
3. This field overlaps with other areas of biology and chemistry, particularly
genetics and biochemistry.
It is the joining of aspects between genetics and biochemistry
Biochemistry is the study of molecules (e.g. proteins). Biochemists take an
organism or cell and dissect it into its molecular components, such as enzymes,
lipids and DNA, and reconstitute them in test tubes (in vitro).
Genetics is the study of the effect of genetic differences on organisms.
4. Overview of Organizations of Life
Nucleus = library
Chromosomes = bookshelves
Genes = books
Almost every cell in an organism contains the same libraries and the same
sets of books.
Books represent all the information (DNA) that every cell in the body needs
so it can grow and carry out its various functions
~3.2 billion base pairs in every cell build the
human genome
Genes form only 1.5% of the human genome
A gene is a segment of the DNA, that encodes
the construction plan for a protein
In humans there are 30,000 genes only
5.
6. A Brief History of molecular biology – A Journey
1865 Gregor Mendel discover the basic rules of
heredity of garden pea.
An individual organism has two alternative
heredity units for a given trait (dominant trait vs.
recessive trait)
1869 Johann Friedrich Miescher discovered DNA and
named it nuclein.
Mendel: The Father of
Genetics
Johann Miescher
1881 Edward Zacharias showed chromosomes are
composed of nuclein.
1899 Richard Altmann renamed nuclein to nucleic acid.
By 1900, chemical structures of all 20 amino acids had
been identified
1902 – Emil Fischer wins Nobel prize: showed amino
acids are linked and form proteins Emil Fischer
7. 1911 – Thomas Hunt Morgan discovers genes on
chromosomes are the discrete units of heredity
1911 - Pheobus Aaron Theodore Lerene
discovers RNA
1941 – George Beadle and Edward Tatum
identify that genes make proteins
1950 – Edwin Chargaff find Cytosine
complements Guanine and Adenine complements
Thymine.
1950s – Mahlon Bush Hoagland first to isolate
tRNA
1952 – Alfred Hershey and Martha Chase make
genes from DNA
Thomas
Morgan
Edwin
Chargaff
Mahlon
Hoagland
8. 1952-1953 - James Watson and Francis Crick deduced
the double helical structure of DNA
1956 - George Emil Palade showed the site of enzymes
manufacturing in the cytoplasm is made on RNA
organelles called ribosomes.
1970 - Howard Temin and David Baltimore
independently isolate the first restriction enzyme
This means that DNA can be cut into reproducible
pieces at specific site by restriction enzymes called
endonuclease
The pieces can be linked to bacterial vectors and
introduced into bacterial hosts.
This is called (gene cloning or recombinant DNA
technology)
James Watson and
Francis Crick
9. 1977 - Phillip Sharp and Richard Roberts demonstrated that
pre-mRNA is processed by the excision of introns and exons are
spliced together.
1986 - Leroy Hood developed automated sequencing mechanism
1986 - Human Genome Initiative announced
1995- Moderate-resolution maps of chromosomes 3, 11, 12, and 22
were published
These maps provide the locations of “markers” on each
chromosome to make locating genes easier
1995 - John Craig Venter: First bacterial genomes sequenced
1995 - Automated fluorescent sequencing instruments and
robotic operations
10. 1996 - First eukaryotic genome-yeast-sequenced
1999 - First human chromosome (number 22) sequenced
2001 - International Human Genome Sequencing published
the first draft of the sequence of the human genome
April 2003 - Human Genome Project Completed & Mouse
genome is sequenced.
April 2004 - Rat genome sequenced.
Next-generation sequencing – genomes being sequenced by the
dozen
Is it the end? Of course not!!
11. Molecular biology
The study of gene structure and function of organisms at the molecular level
The study of molecular basic of the process of replication, transcription and
translation of the genetic material.
The four main families of biochemical molecules in cells :
Carbohydrates Lipid (fats and oils)
Proteins Nucleic acid (DNA and RAN)
1. Nucleic Acids and Nucleotides
Nucleic acids
are macromolecules (DNA and RNA)
central to the storage and transmission of genetic information.
Nucleotides are the subunits (monomers) of DNA and RNA.
Essential for all known forms of life (reproduction)
Functions of nucleic acid
Convey energy (e.g. ATP, GTP),
Encoding, transmitting and expressing genetic information
They are part of essential coenzymes, and
They regulate numerous metabolic functions (enzymes, RNA).
12. Continued……
Nucleotides are composed of:
a phosphate group
a pentose sugar
o Ribose (RNA)
o Deoxyribose (DNA)
nitrogen containing bases
purine- two rings
• Adenine (A)
• Guanine (G)
pyrimidine – contains one ring
• Cytosine (C)
• Uracil (U)
• Thymine (T)
DNA: A,G,C,T
RNA: A,G,C,U
Thymine(T) is a 5-methyluracil (U)
13. Nucleosides and nucleotides
A nucleoside is a compound of a sugar residue (ribose/ deoxyribose) and a base.
The Nitrogen Bases are covalently attached to the 1’ position of a pentose sugar
ring, to form a Nucleoside.
N-glycosidic bond
bond that hold sugar with base together.
formed between the C atom in position 1 of the sugar & an Nitrogen atom of
the base.
NB. Adenosine, Guanosine, Cytidine, Thymidine, Uridine
14. Nucleotides = Nucleoside + Phosphate
A nucleotide is a nucleoside with one or more phosphate groups bound covalently
to the 5’-Carbon of Ribose/ deoxy Ribose. Maximum three phosphates can be
attached.
Deoxynucleotides
(containing deoxyribose
Ribonucleotides
(containing ribose)
15.
16. NITROGEN
BASES
NUCLEOSIDES NUCLEOTIDES
Adenine (A) Adenosine Adenosine 5’-monophosphate (AMP) or Adenylate
Deoxyadenosine Deoxyadenosine 5’-monphosphate (dAMP) or
deoxy Adenylate
Guanine (G) Guanosine Guanosine 5’-monphosphate (GMP)
or Gunaylate
Deoxyguanosine Deoxy-guanosine 5’-monphosphate (dGMP) or
deoxy Guanylate
Cytosine (C) Cytidine Cytidine 5’-monphosphate (CMP)
Cytidylate
Deoxycytidine Deoxy-cytidine 5’-monphosphate (dCMP) or deoxy
Cytidylate
Uracil (U) Uridine Uridine 5’-monphosphate (UMP) or Uridylate
Thymine (T) Thymidine/
Deoxythymidie
Thymidine/deoxythymidie
5’-monphosphate (dTMP) or deoxy Thymidylate
17.
18. Some important nucleotides
dATP, dGTP, dCTP, dTTP
Raw materials for DNA biosynthesis.
ATP, GTP, CTP, UTP
Raw materials for RNA biosynthesis
Energy donor
Important co-enzymes
Cycling nucleotides - cAMP, cGMP
Secondary messengers for some Hormoneses
19. Nucleic acid - DNA & RNA
consists of a series of nucleotides (polynucleotides).
A phosphodiester bridge between the 3’ C atom of one nucleotide and the 5’ C
atom of the next joins two nucleotides
The linear sequence is usually given in the 5’ to 3’ direction
20.
21. DNA structure and properties
DNA is complex polymeric chemical compound which contains four kinds of
smaller building blocks called deoxyribonucleotides.
The base sequence (or the nucleotide sequence) in polydeoxynucleotide chain.
Each deoxyribonucleotide is made up of:
a phosphoric acid molecule (biologically called phosphate)
a pentose sugar called deoxyribose and
pyrimidine and purine nitrogenous bases
22. Continued…
Four major kinds of nitrogenous bases in deoxyribonucleotides of DNA:
two are heterocyclic and two-ringed purines, adenine (A) &guanine (G),
two are one - ringed pyrimidine, cytosine (C) and thymine (T)
Table: Four nitrogen containing bases, nucleosides and nucleotides of DNA
23. Continued…
The four deoxyribonucleotides occur also in nucleoplasm and cytoplasm in their
triphosphate known as:
deoxyadenosine triphosphate (dATP),
deoxyguanosine triphosphate (dGTP),
deoxycytidine triphosphate (dCTP) and
thymidine triphosphate (TTP).
Why do nucleotides occur in nucleotides triphosphates?
Ans - b/c the DNA polymerase enzyme can act only on triphosphate of
deoxyribonucleotides during DNA replication.
Molar Ratios of Nitrogen Bases in DNA Molecule
Edwin Chargaff – discovered that the four nucleotide bases are not necessarily
present in DNA in exactly equal proportions.
He disproved tetranucleotide theory which considered DNA as a monotonous
polymer having four DNA bases in approximately equal molar proportions.
24. Continued………
Chargaff (in 1950) discovered the equivalence rule which suggested that:
the total amount of purines equal to the total amount of
pyrimidines (A+G=T+C)
the amount of adenine equal to the amount of thymine (A=T) and
the amount of guanine equal to the amount of cytosine (G=C).
Chargaff’s equivalence rule is almost universal in different organisms (viruses,
bacteria, plants and animals). E.g.
Higher plants and animals rich A&T & relatively poor in G&C
e.g., AT/GC ratio of DNA of man was 1.40:1
Microorganisms and lower plants and animals rich in G&C &
relatively poor in A & T
e.g., AT/GC ratio of DNA of Mycobacterium tuberculosis
was 0.60 : 1).
Note – Generally percentage of GC content in lower organisms is higher than
that of higher organisms.
25. Watson-Crick Structural Model of DNA
Watson and Crick developed the definitive model of DNA structure.
The adjacent deoxyribonucleotides are joined in a chain by phosphodiester bonds
polynucleotide
5'→3' link b/n consecutive carbon of deoxyriboses
26. Continued…
A DNA molecule :
Two polynucleotides chain
Double helix (like a ladder)
Wrapped (twisted) around each other
Upright strand (backbone)- sugar – phosphate (outside)
Rungs of the ladder – bases (inside)
The two polynucleotides held together- hydrogen bond
The two strands run in anti-parallel, having opposite directions
27. Continued…
DNA is a directional molecule
The complementary strands run in opposite directions
One strand runs 3’-5’
The other strand runs 5’ to 3’ ( the end of the 5’ has
the phosphates attached, while the 3’ end has a
hydroxyl exposed)
DNA helix has two external grooves:
major groove (a deep & wide ) and
minor groove (a shallow & narrow )
Both of these grooves are large enough to allow protein
molecules to come in contact with the bases.
Polymorphism of DNA Helix
DNA was thought to have the same monotonous structure (for about 20yrs):
exactly 36º of helical twist between its adjacent base pairs
10 nucleotide pairs per helical turn and
uniform helix geometry
28. Continued…
The polymorphism of DNA helix can be classified into A-, B-, & Z-DNA (forms)
based on:
number of residues (monomers) per turn (“n”)
the spacing of residues along the helical axis (“h”).
30. DNA Replication
Heterocatalytic function
Directs the synthesis of other
molecules, RNA and protein
Autocatalytic functions
Directs the synthesis of DNA
itself
Why does DNA self-replicate or autocatalytic? Because of:
the specificity of base pairing,
the sequence of base along one chain automatically determines (directs)
the base sequence along the other.
The mechanism: Strand separation, unwinding , followed by copying of each strand.
Each separated strand acts as a template for the synthesis of a new complementary
strand.
31. Models for DNA replication
1) Semiconservative model
Daughter DNA molecules contain
one parental strand and one newly-
replicated strand.
2) Conservative model
Parent strands transfer information
to an intermediate & the
intermediate gets copied.
The parent helix is conserved, the
daughter helix is completely new
3) Dispersive model
Parent helix is broken into
fragments, dispersed, copied then
assembled into two new helices.
New and old DNA are completely
dispersed.
32. Enzymes of DNA replication
Three main enzymes act on DNA in both prokaryotic and eukaryotic cells are:
Nucleases
Polymerase
Ligase
І. Nuclease enzymes
hydrolyze or break down a polynucleotide chain into its component nucleotides
will attack either the 3´ or the 5´ end of this linkage.
digest phosphodiester backbone
There are two types nuclease enzymes:- exonuclease and endonuclease.
a). Exonuclease
begin its attack from a free end of a polynucleotide
It begins at a free 3’-OH end or at a free 5'-Phosphate end (5'→3’)
It travels along the strand & digest the entire polymer ( liberates NMP)
B). Endonuclease
digest phosphodiester bond within the interior of a polynucleotide chain
Single strand-cut the chain into two pieces, double helix, it forms nick
33. II) Polymerase /replicase enzymes
Catalyze the synthesis of one polynucleotide chain that is a copy of another.
In vitro DNA polymerization
Biologists understood the DNA replication in living cells(in vivo) tried in vitro
polymerization using the ff 3 molecules in addition to DNA polymerase:
deoxynucleotide triphosphates ( dATP, dCTP, dGTP and (d) TTP),
Primer: single-stranded DNA (ssDNA)/ RNA polynucleotide chains with free
3'-OH ends
Primers cannot initiate the de novo (the synthesis of complex
molecules from simple molecules) synthesis of a new strand
DNA template – strand that directs polymerization
The DNA polymerase observe the rule of complementary base pairing.
If a free 3'-OH on a primer strand lies opposite a thymine on a template strand:
a polymerase enzyme will add only an adenine group to the primer
Formation phosphodiester bond with a free 3'-OH
a molecule of pyrophosphate (P~P) is simultaneously released.
The hydrolyzes of pyrophosphate into 2 phosphates release energy that drives
polymerization.
36. Prokaryotic DNA polymerases
Three d/t types of DNA polymerase in prokaryotes (E. coli)
a). DNA polymerase I
was 1st isolated in 1960 by Arthur Kornberg
suggested to be involved in DNA replication
now considered to be a DNA repair rather than a
replication enzyme
mainly involved in removing RNA primers from Okazaki
fragments
5’ → 3’ exonuclease activity & polymerization.
b). DNA polymerase IІ - DNA repair enzyme.
c). DNA polymerase IІІ - plays an essential role in DNA replication
a multimeric enzyme or holoenzyme having 10 subunits
(alpha (α), beta (β), epsilon (ε), theta (θ), tau (τ), gamma (γ), delta (δ), delta
dash (δ´), chi (χ), and psi (ψ)
The core enzyme comprises three subunits α, β and θ)
The seven subunits increase processivity (rapidity & efficiency)
37. Eukaryotic DNA polymerases
There are 5 types of eukaryotic DNA polymerases:
DNA polymerase α (alpha) - used as primase (formation of primer).
DNA polymerase β (beta) - concerned with DNA repair.
DNA polymerase γ (gamma) - mitochondrial polymerase and replication
of DNA in mitochondria.
DNA polymerase δ (delta) – polymerization of leading strand and
okazaki fragments
DNA polymerase ε ( epsilon) - DNA repair
(III) DNA ligase
Ligate (bind) a nick of DNA which is created by endonucleases enzyme / removal
of primer.
catalyzing phosphodiester bond formation between free 3´-OH and free 5´-P
groups
ligase enzyme from E.coli requires a cofactor oxidized nicotinamide adenine
dinucleotide (NAD+)
38. Roles of RNA Primers in DNA Replication
DNA polymerase cannot initiate synthesis of DNA without the availability of a
primer RNA strand.
primer RNA strand - short RNA oligonucleotide segments synthesized by primase
enzyme.
RNA primer is synthesized by copying a particular base sequence from one DNA
strand.
Primer is about 10- 20 nucleotides.
39. Replicons
DNA replication in prokaryotes and eukaryotes is started in specific units called
replicons (origin of replication).
Prokaryotic cells (E. coli) has only one origin of replication – single genome
Eukaryotes have many origin of replication (replicons). Eg. Yeast has 500 OriC.
It is A - T rich regions.
The termination of DNA replication requires the product of tus gene is called
ter binding protein (TBP) which recognizes termination sites.
Table: prokaryotes and eukaryotes replicons
40. Proteins involved in Opening of DNA Helix
The three types of proteins need to open and to expose DNA template for the
DNA polymerase:
1). DNA helicases
are ATP- dependent unwinding enzymes which promote separation of the
two parental strands
establish replication forks that will progressively move away from the origin
of replication.
DNA helicases utilize ATP to move rapidly along a DNA single strand
2). Single strand DNA-binding protein ( SSBP)
it is also called Helix-destabilizing strand protein.
prevent from rewinding of the single DNA strands/ duplexes
bind to exposed DNA strands without covering the bases
It also prevents nuclease enzymes from attacking single strands.
coat and straighten out the regions of single-strand DNA
3). Topoisomerases (DNA gyrases)
relax the supercoil by attaching to the transiently supercoil duplex → nicking one
of the strands and rotating it through the unbroken strand → the nick is then
resealed
41.
42. Continued…
DNA topoisomerase can be viewed as a ‘‘reversible nuclease’’ b/c:
It holds energy released when phosphodiester bond is broken to seal the nick.
Type of topoisomerase
Topoisomerase I - a single-strand breaks or nick.
Topoisomerase II – the double-strand break in the helix.
Replisome and Primosome
Replisome
The association of DNA polymerases and helicases at origin of replication.
Why?
Ans:- increases the speed of helicase 10- fold, prevents helicase from
“running away” from replication fork.
Called replication fork enzymes.
Important for the synthesis of leading and lagging strands in a coordinated fashion.
Primosome
the primase is linked directly to the DNA helicase at replication fork to
form a unit on the lagging strand.
Activate primase 1000-fold (not tightly associated)
It moves with the fork, synthesizing RNA primers as it moves
43. Replication complex in eukaryotes. The lagging strand is shown looped around the
replication complex to demonstrate that all DNA synthesis is in the 5’ to 3’ direction.
Single-strand binding proteins (not shown) are bound to the unpaired, single-stranded
DNA. Other proteins also participate in this complex
44. Mechanism of DNA replication in Prokaryotes
In E. coli, the process of DNA replication involves the following three main
steps:
Initiation of DNA replication
Elongation of DNA chain
Termination of DNA replication
1). Initiation of DNA replication
Initiation comprises three steps:
recognition of the origin of replication (Ori C)
The specific sites at which DNA unwinding and initiation of replication occur
are called origins of replication
A-T rich region
DnaA protein (initiator protein) , ATP dependent protein, opening
promoter the only protein that identify DNA sequence
DnaA protein separate, “melt” opening of DNA duplex to generate a region
of single stranded DNA.
capture of DnaB (helicase):
unwinding of the DNA in the presence of ATP, SSB protein and DNA
gyrase (topoisomerase)
also acts as the activator of primase.
45. Continued…
SSB binding occurs on single stranded regions of the single strand DNA.
DNA covered by single-strand binding proteins is rigid, without bends or
kinks.
The junction between the newly separated template strands and the
un-replicated duplex DNA is known as the replication fork.
46. 2.Elongation of DNA chain
During elongation , the following events occur:
Formation of a replication fork by opening the DNA duplex- DnaB (helicase).
The DNA strand having helicase becomes the lagging strand, Primosomes-
synthesizes multiple primers for lagging strand
single RNA primer for the leading strand
The DNA poll III has to work on the lagging strand but it travels in opposite
direction to which DnaB helicase is bound
Dna-B helicase, primase and DNA poll III work together in strand elongation
47. NB. Synthesis (elongation) of lagging and leading strands takes place by somewhat
different; it is far more complex for lagging strand than for the leading strand.
48. Discontinuous synthesis on lagging strand
On the lagging strand, Primase is taken up from solution and is activated by
helicase (Dna-B) to synthesize a RNA primer (10-20nucleotides long).
DNA poll III on the lagging recognizes RNA primers on strand and synthesis
okazaki fragments.
On completion of the okazaki fragments, the RNA primers are excised by DNA
polymerase I, which then fills the resulting gaps with DNA- gap left
The enzyme DNA ligase forms the phosphodiester bond that links the free 3´ end
of the primer replacement of the 5´ end of the okazaki fragment.
Continuous synthesis on leading strand
The leading strand is primed once on each of the parental strands
DNA poll III causes elongation of the leading strand
Finally DNA pol I and ligase enzymes give final touch to the leading strand as in
case of the lagging strand.
51. 3.Termination of DNA Replication
DNA replication terminates when another replication fork or the telomere is
reached.
Termination is mediated by DNA binding proteins called Replication Terminator
Proteins (TUS).
52. DNA Replication in Eukaryotes
DNA replication in both prokaryotes and eukaryotes uses a semiconservative mechanism
and employs leading- and lagging-strand synthesis.
So the components of the replisome in prokaryotes & eukaryotes are very similar.
As organisms increase in complexity, the number of replisome components also increases
dramatically.
One reason for the added complexity of the eukaryotic replisome is the higher complexity
of the eukaryotic template genome.
Unlike the bacterial chromosome, eukaryotic chromosomes exist in the nucleus as
chromatin, the basic unit of chromatin is the nucleosome, which consists of DNA wrapped
around Histone proteins.
Thus, the replisome has to not only copy the parental strands, but also disassemble the
nucleosomes in the parental strands & reassemble them in the daughter molecules.
Eukaryotic DNA replication requires:-
two different DNA polymerase enzymes (DNA polymerase α and DNA polymerase δ).
DNA polymerase δ synthesizes the DNA on the leading strand (continuous DNA
synthesis), whereas DNA polymerase α synthesizes the DNA on the lagging strand
(discontinuous DNA synthesis).
Besides these two enzymes, six more factors are involved in eukaryotic DNA
replication: (1) T antigen; (2) replication factor A or RF-A (also called eukaryotic SSB);
(3) topoisomerase I; (4) topoisomerase II; (5) proliferating - cell nuclear antigen
(PCNA) also called cyclin, and (6) replication factor C or RF-C
53. Continued…
Approximately yeast has 400 (25 each) replication origins are dispersed
throughout the 16 chromosomes of yeast, & there are estimated to be thousands
of growing forks in the 23 chromosomes of humans.
Unwinding of DNA complex- T-antigen (Tumour antigen), RF-A and
topoisomerases I and II binds to Ori.
T-antigen, multi-subunits ( DNA-helicase) local unwinding in the presence of
ATP.
More extensive duplex unwinding occurs due to association of RF-A (SSB)
and a topoisomerase (help in unwinding of DNA by altering topology of DNA
at the replication fork.)
The primer RNA synthesis is performed by primase which is tightly associated
with DNA polymerase α.
DNA polymerase α helps in synthesis of an okazaki fragment in 5' to 3'
direction.
Replication factor C (or RF-C) and PCNA (cyclin) help in switching of DNA
polymerases so that pol α is replaced by pol δ which then continuously
synthesized DNA on the leading strand.
54. Continued…
Another okazaki fragment is then synthesized from the replication fork on the
lagging strand by pol α - primase complex and this step is repeated again and
again, till the entire DNA molecule is covered.
The RNA primers are removed and the gaps are filled as in prokaryotic DNA
replication.
55. Summary of DNA replication, major elements:
Segments of single-stranded DNA are called template strands.
Topoisomerase or DNA gyrase relaxes the supercoiled DNA.
Initiator proteins (T- antigen) and DNA helicase binds to the DNA at the replication
fork and untwist the DNA using energy derived from ATP.
DNA primase next binds to helicase producing a complex called a primosome
Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA
polymerase III or α /δ adds nucleotides.
Polymerase III or α / δ adds nucleotides 5’ to 3’ on both strands beginning at the
RNA primer.
The RNA primer is removed and replaced with DNA by polymerase I or polymerase
ε , and the gap is sealed with DNA ligase.
Single-stranded DNA-binding (SSB) proteins (>200) or RFA (Replication Factors A)
stabilize the single-stranded template DNA during the process.
56. DNA MUTATION AND GENOME REPAIR
A number of complex machineries have evolved for recognizing and correcting the
different types of damage caused to DNA.
A mutation is a heritable change in DNA structure (base sequence is changed)
The main causes of DNA mutation are:
Through mistakes during DNA replication
Spontaneous mutations (deamination, depurination that occur
naturally)
Induced mutations caused by environmental agents (chemical
mutagens, UV-radiation)
Mutations are classified into several “types”
point mutation - change of a base in DNA sequence
Deamination – removal amino group from Cytosine – uracil
polymerase slippage during DNA replication - the addition of extra
bases in a ‘microsatellite’ sequence.
Depurination- removal of purine (A &G) from ribose backbone.
- leading to loss of the ribose and a break in the DNA strand
Thymine dimer - cross-linking of bases b/n thymines
57. Mechanisms of DNA Repair
A fundamental difference from RNA, protein, and lipid
All these others can be replaced, but DNA must be preserved
How do the mutations get repaired??
E. coli is the model system for understanding different repair mechanisms
1). Direct repair
Repairs damage without removing the damaged base
Pyrimidine dimers can be repaired by DNA photolyase enzyme!
Simple nicks in one of the phosphodiester backbones are corrected by the
enzyme DNA ligase
2). Excision repair
a). Base Excision Repair - the damaged base is excised first by a DNA glycosidase
-Endonucleases then cleave the sugar-phosphate backbone,
leaving a single base gap, which is repaired by DNA polymerase
b). Nucleotide Excision Repair
the entire nucleotide(s) and a short region around them are removed by
an endonuclease complex.
the gap created is filled in by DNA polymerase and the remaining nick
sealed by DNA ligase.
58. Continued…
3). Mismatch repair - scan newly-replicated DNA duplexes for mismatched bases
DNA polymerases are extremely accurate at copying DNA templates (10-7 error
rate for E. coli) but they are not perfect.
This great accuracy involves a mechanism called proofreading.
The major DNA polymerases of prokaryotes and eukaryotes possess a 3’–5’
exonuclease activity, which removes any nucleotide that has not been correctly
base paired with the template during the extension reaction .
But rarely an incorrect nucleotide is inserted into a new DNA strand by DNA
polymerase, which is corrected by mismatch repair
A short region on one of the two strands must be removed and the lesion filled in
by DNA polymerase І.
How does the repair machinery know which strand to remove? DNA methylation
distinguishes old DNA from newly synthesized DNA.
4). Repairing Double-Stranded DNA Breaks - extremely dangerous for the cell
If such damage is not repaired the exposed ends might become degraded, leading
to a deletion of DNA & lose of an entire chromosome.
In humans double-stranded DNA breaks are repaired by DNA ligase in concert
with a multi-subunit complex containing DNA protein kinase (bring break ends)
59. RIBONUCLEIC ACID (RNA)
Some plant viruses (TMV), animal viruses (e.g., influenza viruses, poliomyelitis
viruses, etc.) and bacteriophages (e.g., MS2) store genetic information on RNA.
RNA is a polymer made up of monomeric nucleotide units (Ribonucleotides)
Each ribonucleotide contains:
a pentose sugar (ribose)
a molecule of phosphate group and
a nitrogen base.
The nitrogen bases of RNA are two purines(A&G) and two pyrimidines (C&U)
The four ribonucleotides also occur freely in nucleoplasm in the form of
triphosphates of ribonucleosides such as adenosine triphosphate (ATP), and
uridine triphosphate (UTP) , cytidine triphosphate(CTP) & GTP (guanosine
triphospahte ).
60. Molecular Structure of RNA
RNA molecule may be either single stranded or double stranded but not helical.
Each strand of RNA is polynucleotide, made up of many ribonucleotides.
In the polynucleotide strand of RNA, the ribose and phosphoric acids of
nucleotides remain linked by phosphodiester bonds.
RNA can be classified into:
Genetic RNA – RNA that used to store genetic information, no DNA
Non-genetic RNA- use the RNA in carrying the orders of DNA and have
no genetic role, have DNA
e.g. rRNA, mRNA, tRNA, and snRNA
61. Non-Genetic Ribonucleic Acid (RNA) and Transcription
A DNA molecule does not leave the nucleus to participate directly in
the process of protein synthesis
It uses RNAs molecules for carrying its genetic information’s from
the nucleus to the site of protein synthesis (ribosomes).
The transfer of genetic information from DNA to RNA molecules
and then from RNA to protein molecules is called gene expression.
RNA molecules are synthesized by using the base sequences on one
strand of DNA as a template by enzymes called RNA polymerases.
The process by which RNA molecules are initiated, elongated, and
terminated is called transcription.
62. Comparison between DNA replication and transcription
DNA Replication
DNA-dependent DNA polymerase
Thymine
Identical copies of two daughters DNA
molecules
The new DNA strand remains
bounded to the template DNA
One or few initiation points for DNA
replication (large portions of genome
are copied into single)
to conserve the entire genome for
next generation
Repaired incase of damage
Committed the least error
Single function
DNA Polymerase needs primer
DNA Transcription
DNA-dependent RNA polymerase
Uracil
Single strand that have opposite
polarity to the template DNA.
The RNA transcripts do not ordinarily
remain hydrogen-bonded with their
DNA templates
Transcription is initiated at close
intervals along the DNA ( relatively
short RNA copies are produced)
To make RNA copies of individual
genes that the cell can use.
Degraded after specific function
Many functions
RNA polymerase doesn’t need primer
63. Types of RNAs (Non-Genetic)
Based on their specific functions during the process of protein synthesis, the
following non-genetic RNA molecules have been recognized in cells:
1) Ribosomal RNA (rRNA)
stable or insoluble RNA
the most abundant (up to 80%) of the total cellular RNA
Structural and functional components of ribosomes
Types - 28S rRNA, 18S rRNA, 5.8 S and 5S rRNA.
Subunits – 80s (eukaryotes), & 70s (prokaryotes)
2) Messenger RNA (mRNA)
the largest, but the least abundant( 5%)
base sequence complementary to DNA
carry DNA’s genetic information’s for the assembly of amino acids into the
polypeptide.
3) Transfer RNA (tRNA)
about 15% tRNA
the smallest
Important in translation
64. RNA polymerase
In any gene only one DNA strand acts as the template for transcription.
The sequence of nucleotides in RNA depends on their sequence in the DNA
template.
The bases T, A, G, and C in the DNA template will specify the bases A, U, C, and
G, respectively, in RNA. DNA is transcribed into RNA by the enzyme RNA
polymerase.
Transcription requires this enzyme to recognize the beginning of the gene to be
transcribed and catalyze the formation of phosphodiester links between
nucleotides that have been selected according to the sequence within the DNA
template.
RNA polymerase is one of the largest enzymes known (MW 490,000).
E. coli (prokaryotic) RNA Polymerase consists of five subunits:
two identical alpha (α) subunits and
one chain of each of beta (β), beta dash (β´) – catalytic activity and
unwinding of DNA.
sigma (δ) subunits (not tightly bind) – recognition of promoter on DNA
and directs RNA polymerase in selecting the initiation sites (promoter).
65. Mechanism of prokaryotic transcription
Escherichia coli genes are all transcribed by the same RNA polymerase. This
enzyme is made up of α, α’, β,β’ and σ . Each of the subunits has its own job to do
in transcription.
The role of the sigma ( σ ) factor is to recognize a specific DNA sequence called
the promoter, which lies just upstream of the gene to be transcribed.
A promoter is the DNA sequence that initially binds the RNA polymerase (together
with an initiation factors (σ)).
The nucleotide in the template strand at which transcription begins is designated
with the number +1.
Transcription proceeds in the downstream direction, and nucleotides in the
transcribed DNA are given successive positive numbers (to the right of the
transcription start site).
Nucleotides that lie to the left of this site are called the upstream sequences and
are identified by negative numbers (to the left).
66. Continued…
E. coli promoters contain two important regions. One centered around nucleotide
−10 (i.e., 10 bases before the transcription start site) usually has the sequence
TATATT. This sequence is called the the Pribnow box.
The second, centered near nucleotide−35 often has the sequence TTGACA. This is
the−35 box (i.e., 35 bases before the transcription start site).
This region is generally referred to as the TATA box and is believed to orient the
RNA polymerase enzyme, so that synthesis proceeds from left to right.
The sigma factor (σ) subunit first binds to the –35 sequence in a highly specific
interaction and then, the appropriate region of this huge enzyme can come in
contact with the –10 sequence of Pribnow box.
On binding to the promoter sequence the σ (sigma factor) brings the other subunits
(two of α plus one each of β and β’) of RNA polymerase into contact with the
DNA to be transcribed. This forms the closed promoter complex.
67. Continued…
For transcription to begin, the two strands of DNA must separate, enabling one
strand to act as the template for the synthesis of an RNA molecule. This formation
is called the open promoter complex.
The separation of the two DNA strands is helped by the AT-rich sequence of the
−10 box.
There are only two hydrogen bonds between the bases adenine and thymine; thus it
is relatively easy to separate the two strands at this point.
DNA unwinds and rewinds as RNA polymerase advances along the double helix,
synthesizing an RNA chain as it goes. This produces a transcription bubble.
Once an open-promoter complex has formed, RNA polymerase is ready to initiate
RNA synthesis.
First RNA polymerase binds to purine triphosphates and usually ATP is the first
nucleotide added in the growing RNA chain. Thus, the first DNA base that is
transcribed is usually thymine (T).
The RNA chain grows in the 5’ to 3’ direction, and the template strand is read in
the 3’ to 5’ direction.
68. Continued…
When the RNA chain is about 10 bases long, the σ factor is released from RNA
polymerase and plays no further role in transcription.
The β subunit of RNA polymerase binds ribonucleotides and joins them together
by catalyzing the formation of phosphodiester links as it moves along the DNA
template.
The β’ subunit helps to keep the RNA polymerase attached to DNA.
The two α subunits are important as they help RNA polymerase to assemble on
the promoter.
69. Continued…
The elongation site is then filled with a ribonucleotide triphosphate that is
selected strictly by its ability to form a hydrogen bond with the next base in the
DNA strand.
The elongation phase begins when the RNA polymerase releases the base and
then moves along the DNA chain.
During elongation phase addition of 40 bases-per second at 37ºC takes place.
The open region extends only over a few base pairs; that is, the DNA helix
recloses (rewinds) just behind the enzyme.
The newly synthesized RNA is released from its hydrogen bonds with the DNA
as the helix reforms.
70. Chain (transcription) Termination
Termination of RNA synthesis (or transcription) occurs at specific base sequences
in the DNA molecule.
RNA polymerase has to know when it has reached the end of a gene. E. coli has
specific sequences, called terminators, at the ends of its genes that cause RNA
polymerase to stop transcribing DNA.
Terminators trigger the elongating polymerase to dissociate from the DNA and
release the RNA chain it has made.
In bacteria, terminators come in two types: Rho -dependent and Rho-independent.
1) Rho –dependent terminator
requires a protein called Rho to induce termination.
Rho, which is a ring-shaped protein with six identical subunits, binds to single-
stranded RNA as it exits the polymerase.
The protein also has an ATPase activity, and once attached to the transcript,
Rho uses, the energy derived from ATP hydrolysis to induce termination.
Rho pulls RNA out of the polymerase, resulting in termination
It induces a conformational change in polymerase, causing the enzyme to
terminate.
72. 2. Rho-independent terminator
Also called intrinsic terminators because they need no other factors
(proteins& ATP) to work.
A terminator sequence consists of two regions rich in the bases G and
C that are separated by about 10 bp. This sequence is followed by a
stretch of A bases.
When the GC-rich regions are transcribed, a hairpin loop forms in the
RNA with the first and second GC-rich by base-pairing with itself.
Formation of the hairpin causes termination by disrupting the
elongation complex and causes the transcription bubble to shrink .
The remaining interactions between the adenines in the DNA template
and the uracils in the RNA chain have only two hydrogen bonds per
base pair and are therefore too weak to maintain the transcription
bubble.
73.
74. Mechanism of eukaryotic transcription
Eukaryotes have three types of RNA polymerase. Those are:
RNA polymerase I - transcribes the genes that code for most of the
ribosomal RNAs (rRNAs).
RNA polymerase II - transcribes all messenger RNAs (mRNAs)
RNA polymerase III- transcribes transfer RNA (tRNAs) genes and also
catalyzes the synthesis of several small RNAs including the 5S rRNA.
Unlike bacteria, several initiation factors are required for efficient and promoter-
specific initiation in eukaryotes. These are called the transcription factors (TFs).
The nature and function of RNA polymerase II (for mRNA) is well studied than
other two types of RNA polymerases.
Eukaryotic promoters contain three distinct regions lying between – 25 bp and -
100 bp. Those are:
TATA-box or Hogness box
is located 20 bp upstream to the starting point
7bp long
aligns RNA polymerase at proper site with the help of proteins, called
transcription factors or TFs.
75. Continued…
CAAT box (or CAT box)-
lies between –70 and –80 base pairs
includes GGT/ACAATCT base composition
necessary for initiation, is conserved in some promoters
GC box (GGGCGG)
is found in one or more copies at –60 or –100 bp upstream
determine the efficiency of transcription with CAT box
Eukaryotic promoters also consist of sites located 100 to 200 base pairs
upstream, which interact with proteins other than RNA polymerase and, thus,
regulate the activity of promoter. These sites are called enhancers.
Enhancers increase in the rate of transcription up to 200 fold.
There are other regulatory sites known as silencers which repress gene
expression.
Both enhancers and silencers can function at great distance (often many
kilobases) from the genes they enhance and repress respectively.
76.
77. A. Initiation of Eukaryotic Transcription
For the eukaryotic transcription:
the regulatory DNA sequences ( promoters, enhancers and silencers) for
genes transcribed by each of the three RNA polymerases differ.
Various transcription factors(TFs) are required for initiation of
transcription ( formation of transcription complex)
TFs is not part of RNA polymerase, help in DNA binding of RNA Pol
B. Elongation of RNA Chain in Eukaryotes
Elongation factors (accessory protein), which enhance the overall activity of
RNA polymerase II and lead to increase in the elongation rate.
EFs helping in the forward movement of RNA polymerase.
C. Termination of Eukaryotic Transcription
In eukaryotes, the actual termination of RNA polymerase II activity during
transcription may take place through termination sites similar to those found in
prokaryotes.
78. Post-transcriptional modification of mRNA
Translation closely follows transcription in prokaryotes.
In eukaryotes, these processes are separated - transcription in nucleus,
translation in cytoplasm
On the way from nucleus to cytoplasm, the mRNA is converted from a
"primary transcript" to "mature mRNA"
The primary transcript may range from 500 to 50,000 nucleotides; it remains
confined to the nucleus and is called heterogeneous nuclear RNA (hnRNA).
The primary transcript of mRNA molecules undergo mRNA processing which
includes the following steps:
5’ end = capping
3’ end = polyadenylation
internal = splicing
1. Addition of a cap of 7-MeG or m7G
During capping process, a cap of a methylated guanosine, called 7-
methylguanosine (7-MeG or m7G), is added to 5' end of primary transcript in a
rare 5'-5' linkage.
81. Why have a Cap?
The CAP structure promotes stability of the mRNA (prevents degradation by 5’
exonucleases) and promotes translation of the mRNA
A ribosomal protein subunit interacts with the CAP and recruits mRNA to the
ribosome.
Untranslated regions (UTR). The mature mRNA has some base pairs at 5'- as well
as 3'-ends that do not code for amino acid sequence of the target protein
2. Addition of tail of poly-A
A poly(A) tail is added to the 3’ end of the transcript in 2 steps:
1) cleavage: the RNA is cut 10-30 nucleotides downstream of a specific
sequence in the 3’UTR
2) addition of A’s (100-200 are added) to generate a poly(A) tail
Why are only mRNAs Capped and have a Poly(A) tail?
The CAP structure is only added to transcripts synthesized by RNAP II
(i.e. mRNA transcripts)
This is because the capping enzymes bind to a part of RNAP II that is
unique to that enzyme (not found in RNA Pol I or RNA Pol III)
82. 3.RNA splicing
The transcript (mRNA) contains intervening sequences (introns) between the
exons that carry the coding information.
Splicing is the process of by which the introns are removed and the exons are
joined together to produce the matured mRNA.
The splicing of RNA is performed by complex proteins called spliceosome.
83. Protein Synthesis (Translation)
The genetic code on mRNA dictates the sequence of amino acids in a protein
molecule.
The synthesis of proteins is quite complex, requiring three types of RNA(mRNA,
tRNA & rRNA (ribosome)
Messenger RNA (mRNA) contains the code and is the template for protein
synthesis.
Transfer RNAs (tRNAs) are adapter molecules that carry amino acids to the
mRNA.
Ribosomal RNAs (rRNAs) form part of the ribosome (moving protein-
synthesizing machines) that brings together all the components necessary for
protein synthesis.
Several enzymes also help in the construction of new protein molecules.
A ribosome attaches near the 5’ end of an mRNA strand and moves toward the 3’
end, translating the codons to polypeptide chain as it goes.
Synthesis begins at the amino end of the protein, and the protein is elongated by
the addition of new amino acids to the carboxyl end.
85. Continued…
Protein synthesis can be divided into four stages:
the binding of amino acids to the tRNAs
Initiation- the components necessary for translation are assembled at the
ribosome;
Elongation- amino acids are joined, one at a time, to the growing
polypeptide chain; and
Termination- protein synthesis stops at the stop codon ( UAG, UAA, &
UGA) and the translation components are released from the ribosome.
1. The Binding of Amino Acids to Transfer RNAs
Amino acids are not directly incorporated into protein on a messenger RNA
template
The 1st stage of translation is the binding of tRNA molecules to their appropriate
amino acids.
86. Continued…
The function of tRNA is to ensure that each amino acid incorporated into a protein
corresponds to a particular codon (a group of three consecutive nucleotides) in the
messenger RNA.
The tRNA serves this function through its structure: It has an anticodon at one end
and an amino acid attachment site at the other end.
The “correct” amino acid, corresponding to the anticodon, is attached to the transfer
RNA by enzymes known as aminoacyl-tRNA synthetases to 3’- end (-OH).
A transfer RNA with an amino acid attached is said to be “charged /activated
tRNA.”
An aminoacyl-tRNA synthetase joins a specific amino acid to its transfer RNA in a
two-stage reaction that takes place on the surface of the enzyme.
1). 1st , activate the amino acid by reacting with ATP
Amino acid + ATP →Aminoacyl AMP + 2PPi
2). Transfer the activated amino acid to its specific tRNA
Aminoacyl AMP +tRNA → Aminoacyl-tRNA + AMP
87. tRNA structure
A typical clover-leaf model tRNA contains the
following structural components:
amino acid attachment site (acceptor stem)-
All tRNAs have unpaired (single stranded)
CCA sequence at the 3´end.
The D loop takes its name from the
characteristic presence of dihydrouridines in
the loop.
The T-loop- is composed of five paired
bases and the last is C-G. T-loop
contains seven unpaired bases and is
involved in the binding of tRNA
molecules to the ribosomes.
The anticodon loop – a three-
nucleotide long sequence that is
responsible for recognizing the codon
by base pairing with the mRNA.
88. Continued…
Each tRNA is specific for a particular kind of amino acid.
All tRNAs have the sequence CCA at the 3’ end, and the carboxyl group (COO-)
of the amino acid is attached to the 2’- or 3’- hydroxyl group of the adenine
nucleotide at the end of the tRNA.
If each tRNA is specific for a particular amino acid but all amino acids are attached
to the same nucleotide (A) at the 3’ end of a tRNA, how does a tRNA link up with
its appropriate amino acid?
The key to specificity between an amino acid and its tRNA is a set of enzymes
called aminoacyl-tRNA synthetases.
A cell has 20 different aminoacyl-tRNA synthetases, one for each of the 20 amino
acids.
Each synthetase recognizes a particular amino acid, as well as all the tRNAs that
accept that amino acid.
89. THE RIBOSOME
The ribosome is the cell’s factory for protein synthesis.
Each ribosome consists of two subunits, one large and one small, each of which is
made up of rRNA plus a large number of proteins.
The ribosomal subunits and their RNAs are named using a parameter, called the S
value or Svedberg unit ,is a sedimentation rate.
It is a measure of how fast a molecule moves in a gravitational field. For ex, the
bigger a ribosomal subunit, the quicker it will sediment and the larger the S value.
Prokaryotic ribosomes, and those found inside mitochondria and chloroplasts, are
70S when fully assembled and comprise a larger, 50S subunit and a smaller 30S
one.
Eukaryotic ribosomes are 80S when fully assembled and comprise a larger, 60S
subunit and a smaller 40S one.
The formation of a peptide bond between two amino acids takes place on the
ribosome.
90. continued…
The ribosome has binding sites for the mRNA
template and for two charged tRNAs.
The ribosome then contains 3 RNA binding sites,
designated A, P, & E.
The `A` site binds an aminoacyl-tRNA (a tRNA
bound to an amino acid); the `P` site binds a
peptidyl-tRNA (a tRNA bound to the peptide
being synthesized); & the `E` site binds a free
tRNA before it exits the ribosome.
2. The Initiation of Translation
The main task of initiation is to place the first
aminoacyl- tRNA in the A site of the ribosome.
In most prokaryotes & all eukaryotes, the first
amino acid in any newly synthesized polypeptide
is methionine, specified by the codon AUG.
It is inserted not by tRNAMet but by a special
tRNA called an initiator, symbolized tRNA-Meti.
91. Continued…
Initiation brings together mRNA, a tRNA with the first amino acid, and the
two ribosomal subunits.
First, a small ribosomal subunit binds with mRNA & a special
initiator tRNA, which carries methionine and attaches to the start
codon (AUG).
Initiation factors bring in the large subunit such that the initiator
tRNA occupies the P site.
92. 3. Elongation of translation
It is during the process of elongation that the
ribosome most resembles a factory.
The mRNA acts as a blueprint specifying the
delivery of charged tRNAs, each carrying an
amino acid.
Each amino acid is added to the growing
polypeptide chain while the deacylated tRNA is
recycled by the addition of another amino acid.
Elongation carried out with the involvement of
protein factors called elongation factors.
During peptide bond formation, an rRNA
molecule catalyzes the formation of a peptide bond
between the polypeptide in the P site with the new
amino acid in the A site.
This step separates the tRNA at the P site from the
growing polypeptide chain and transfers the chain,
now one amino acid longer, to the tRNA at the A
site.
93. Continued…
During translocation, the ribosome moves the tRNA with the attached polypeptide
from the A site to the P site. Because:
the anticodon remains bonded to the mRNA codon, the mRNA moves along
with it.
The next codon is now available at the A site.
The tRNA that had been in the P site is moved to the E site and then leaves the
ribosome.
Translocation is fueled by the hydrolysis of GTP.
It ensures that the mRNA is “read” 5’ -> 3’ codon by codon.
95. Termination of translation
The elongation cycle continues until the codon in the site is one of the three stop
codons: UGA, UAA, or UAG.
A STOP codon causes the mRNA-ribosome complex to fall apart b/c no tRNAs
recognize these codons.
Instead, proteins called release factors recognize stop codons( UAA, UAG &
UGA)
A release factor binds to the stop codon and hydrolyzes the bond between the
polypeptide and its tRNA in the P site.
A ribosome requires less than a minute to translate an average-sized mRNA into a
polypeptide.
96. Differences of prokaryotes and eukaryotes translation
Prokaryotes (bacterial) translation
transcription and translation take place
simultaneously in cytoplasm
mRNA in bacterial cells is short lived,
a few minutes
The large subunit of bacterial
ribosome is only one rRNA.
the small subunit of the ribosome
attaches directly to the region
surrounding the start codon through
hydrogen bonding.
Eukaryotes translation
Transcription in nucleus and
translation in cytoplasm separately.
the longevity of mRNA in eukaryotic
cells is highly variable (hours/days)
the large subunit of the eukaryotic
ribosome contains three rRNAs
the small subunit of a eukaryotic
ribosome first binds to proteins
attached to the 5’ cap on mRNA and
then migrates down the mRNA
Similarities of prokaryotic and eukaryotic translation
The genetic code and start codon (AUG) of bacterial and eukaryotic cells is
virtually identical
Aminoacyl-tRNA synthetases attach amino acids to their appropriate tRNAs
Elongation and termination are similar in bacterial and eukaryotic cells.
mRNAs are translated multiple times and are simultaneously attached to several
ribosomes, forming polyribosomes.
97. The Post-translational Modifications of Proteins
After translation, proteins in both prokaryotic and eukaryotic cells may undergo
alterations called posttranslational modifications.
A number of different types of modifications are possible. These are:
Cleavage and trimming of larger precursors proteins by enzymes before the
proteins can become functional.
For others, the attachment of carbohydrates may be required for activation.
The proper folding of the polypeptide chain; some proteins spontaneously
fold into their correct shapes, but, for others, correct folding may initially
require the participation of other molecules called molecular chaperones.
In eukaryotic cells, the amino end of a protein is often acetylated after
translation.
The removal of 15 to 30 amino acids, called the signal sequence, at the
amino end of the protein. The signal sequence helps direct a protein to a
specific location within the cell, after which the sequence is removed by
special enzymes.
Amino acids within a protein may also be modified: phosphates, carboxyl
groups, and methyl groups are added to some amino acids.
98. Translation and Antibiotics
Antibiotics are drugs (chemicals) that kill microorganisms.
To make an effective antibiotic not just any poison will do the trick is to kill the
microbe without harming the patient.
Antibiotics must be carefully chosen so that they destroy bacterial cells but not the
eukaryotic cells of their host.
Translation is frequently the target of antibiotics because translation is essential to
all living organisms and differs significantly between bacterial and eukaryotic
cells.
A number of antibiotics bind selectively to bacterial ribosomes and inhibit various
steps in translation, but they do not affect eukaryotic ribosomes.
Tetracycline- are a class of antibiotics that bind to the A site of bacterial
ribosomes and block the entry of charged tRNAs.
Chloramphenicol -binds to the large subunit of the ribosome and blocks
peptide-bond formation.
Streptomycin - binds to the small subunit of the ribosome and inhibits
initiation.
99. Regulation of Gene Expression
The synthesis of particular gene products is controlled by mechanisms called
gene regulation.
Cells contain the genetic capacity for the synthesis of different proteins, not all
of these products are present at any given time, many being selectively activated
only upon special occasion and in response to some environmental stimulus.
For example, in prokaryotes some enzymes are synthesized continuously,
indicating that transcription of mRNA is constantly occurring in them.
Other enzymes are synthesized only when a need for their action arises, and
when this need has been fulfilled, enzyme synthesis stops.
Investigations have established that regulation of gene activity may occur at
three levels: transcription, translation and post-translation.
The most efficient place to control gene expression is at the level of
transcription.
The gene regulatory systems of prokaryotes and eukaryotes are slightly different
from each other.
100. Transcription Regulation in Prokaryotes
Why is it necessary? Because:
Bacterial environment changes rapidly
Survival depends on ability to adapt
Bacteria must express the enzymes required to survive in that environment
Enzyme synthesis is costly (energetically)
Therefore, bacteria want to make enzymes when needed.
Genes are very often controlled by extracellular signals; in the case of bacteria,
this typically means molecules present in the growth medium.
These signals are communicated to genes by regulatory proteins, which come in
two types: positive regulators/activators and negative regulators/repressors .
Typically, these regulators are DNA-binding proteins that recognize specific sites
at or near the genes they control.
Positive Regulation (activator)
It improves the ability of RNA polymerase to bind to and initiate
transcription at a weak promoter.
increases transcription of the regulated gene
Example :- cAMP (cyclic adenosine monophosphate)
101. Negative Regulation (repressor)
a negative regulatory factor (repressor) that blocks the ability of RNA
polymerase to bind to and initiate transcription.
repressors decrease or eliminate that transcription.
Most activators and repressors act at the level of transcription initiation.
Each bacterial promoter usually controls the transcription of a cluster of genes
coding for proteins that work together on a particular task. This collection of
related genes is called an operon and is transcribed as a single mRNA molecule
called a polycistronic mRNA.
102. Continued…
The whole operon is transcribed, from a single promoter, into one long mRNA
molecule from which each of the proteins is translated separately.
Operons have three functional “parts”
1) structural genes: these encode proteins (usually with related functions)
2) promoter – specific DNA sequences where RNA pol binds to start
transcription.
3)operator - determines whether the genes are expressed or not.
site of binding of a regulatory protein /repressor proteins.
Segment of DNA that acts like an On/Off switch for transcription
positioned within promoter or between promoter & genes
Binding of a repressor protein to the operator region prevents the binding of RNA polymerase
to the promoter and inhibits transcription of the structural genes of the operon
Repressor proteins are encoded by regulatory genes (enhancers or silencers),
which may be located anywhere in the genome.
103. The Operon Model
1). Inducible system
Induction is the process whereby an inducer (a small molecule) stimulates the
transcription of an operon.
The inducer is frequently a sugar (or a metabolite of the sugar), and the proteins
produced from the inducible operon allow the sugar to be metabolized.
The inducer binds to the repressor, inactivating it.
The inactive repressor does not bind to the operator.
RNA polymerase, therefore, can bind to the promoter and transcribe the operon.
The structural proteins encoded by the operon are produced.
2). Repressible system is a system of enzymes whose presence is repressed, stopping
the production of the end product when it is no longer needed.
Repressible systems are repressed by an excess of the end product of their synthetic
(anabolic) pathway.
NB. The best-studied inducible system is the lac operon in E. coli and repressible
system is tryptophan (trp operon).
LAC OPERON (INDUCIBLE SYSTEM)
Lactose Metabolism – by bacteria
Lactose (milk sugar—a disaccharide) is β –galactoside that E. coli can use for
energy & as a carbon source after it is broken down into glucose & galactose.
The enzyme that performs the breakdown of lactose is β –galactosidase.
The enzyme can additionally convert lactose to allolactose is also important
104. Continued...
When the synthesis of β-galactosidase (encoded by the lacZ) is induced, the
production of two additional enzymes : - galactoside permease (encoded by the
lacY) & β-galactoside acetyltransferase (encoded by the lacA ).
Two other proteins that are turned on when this system is induced are: β-galactoside
permease, which is a transport protein to bring lactose into the cell and β-
galactoside-acetyl-transferase, which protects cells from toxic products.
The presence of the lactose molecule permits transcription of the genes of the lac
operon, which act to break down the lactose.
After all the lactose is metabolized, the repressor returns to its original shape & can
again bind to the operator.
The repressor is an allosteric protein(AP), binds with the inducer(allolactose) and
the second molecule is the operator DNA.
When allolactose is bound to the repressor, it causes the repressor to change shape
and simply dissociates from the operator. .
105. Continued…
After the repressor releases from the operator, RNA polymerase can now begin
transcription.
The three lac operon genes are then transcribed & subsequently translated into
their respective proteins.
106. Continued…
The second control mechanism is a response to glucose, which uses the Catabolite
activator protein (CAP) to greatly increase production of β-galactosidase in the
absence of glucose.
Cyclic adenosine monophosphate (cAMP) is a signal molecule whose prevalence
is inversely proportional to that of glucose.
It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site,
which assists the RNA Pol in binding to the DNA.
In the absence of glucose, the cAMP concentration is high & binding of CAP-
cAMP to the DNA significantly increases the production of β-galactosidase,
enabling the cell to hydrolyse (digest) lactose & release galactose & glucose.
107. Gene regulation in eukaryotes
In eukaryotes, gene regulations are performed by different mechanisms. These are:
Transcription is inhibited during cell division – due to condensation of
chromatin.
Transcription also repressed by different enzymes, proteins & ions like
RNA polymerases- short in supply, they tend to affect it.
Endonucleases- by introducing into DNA nicks that may serve as
initiation sites for some polymerases.
Topoisomerases, helicases and other DNA helix-destabilizing
proteins- render transcription.
DNA methylase - likely to make DNA less available for transcription,
and factors that antagonize methylation would enhance transcription.
ATP - influence the transcription rate by changing the available
energy.
Ions and small molecules – ions like calcium, magnesium and
manganese directly affect chromatin conformation, which modulates
gene activity.
