Molecular Biology - PowerPoint Presentation

Slide1

Molecular Biology

Miruka Conrad Ondieki

Department of Biochemistry-KIU(Western Campus)

Slide2

Organisation

of the Genome

Prokaryotic cell’s genome

Eukaryotic cell’s genome

Slide3

Prokaryotic cell’s genome

The DNA of a bacterial cell, such as

Escherichia coli,

is a circular double-stranded molecule often referred to as the bacterial chromosome

In

E. coli

this DNA molecule contains 4.6 million base pairs

The circular DNA is packaged into a region of the cell called the

nucleoid

In the

nucleoid

, the DNA is organized into 50 or so loops or domains that are bound to a central protein scaffold, attached to the cell membrane

In the loops, DNA is negatively

supercoiled

(

twisted upon itself)

It is also complexed with several DNA-binding proteins, the most common of which are proteins

HU, HLP-1

and

H-NS

Slide4

Prokaryotic cell’s genome…

Slide5

Eukaryotic cell’s genome

The genomic DNA of a eukaryotic cell is contained within a specialized organelle, the nucleus

A typical human cell contains 1000 times more DNA than the bacterium

E. coli

This very large amount of eukaryotic nuclear DNA is tightly packaged in chromosomes

With the exception of the sex chromosomes, diploid eukaryotic organisms such as humans have two copies of each chromosome, one inherited from the father and one from the mother

Chromosomes contain both DNA and protein

Most of the protein on a weight basis is

histones

but there are also many thousands of other proteins found in far less abundance

Slide6

Eukaryotic cell’s genome…

The nuclear DNA–protein complex is

calle

chromatin

The mitochondria and chloroplasts of eukaryotic cells also contain DNA but, unlike the nuclear DNA, this consists of double-stranded circular molecules resembling bacterial chromosomes

In the nucleus, each chromosome contains a single linear double-stranded DNA molecule

The extensive packaging of DNA in chromosomes results from three levels of folding involving:

Nucleosomes

30 nm filaments

Radial loops

Slide7

Eukaryotic cell’s genome…

Slide8

Cell cycle

Although cell division occurs in all organisms, it takes place very differently in prokaryotes and eukaryotes

There are two distinct types of eukaryotic cell division:

Mitosis leads to production of cells that are genetically identical to their parent

Meiosis leads to production of cells with half the genetic content of the parent

Mitosis serves as the basis for producing new cells, meiosis as the basis for producing new sexually reproducing organisms.

Slide9

Cell cycle

In a population of dividing cells, whether inside the body or in a culture dish, each cell passes through a series of defined stages, which constitutes the

cell cycle

The cell cycle can be divided into two major phases;

M phase

Interphase

Slide10

M phase

M phase

includes

the process of mitosis, during which duplicated chromosomes are separated into two nuclei

cytokinesis

, during which the entire cell divides into two daughter cells

Whereas M phase usually lasts only an hour or so in mammalian cells,

interphase

may extend for days, weeks, or longer, depending on the cell type and the conditions

Slide11

Interphase

Interphase

,

which is the period between cell divisions, is a time when the cell grows and engages in diverse metabolic activities

Interphase

lasts for days, weeks, or longer, depending on the cell type and the conditions

Numerous preparations for an upcoming mitosis occur during

interphase

, including replication of the cell’s DNA

The period of time between the end of DNA synthesis and the beginning of M phase is termed as G2 (for second gap)

Slide12

S phase

DNA replication occurs during a period of the cell cycle termed

S phase

S phase is also the period when the cell synthesizes the additional histones that will be needed as the cell doubles the number of nucleosomes in its chromosomes

Slide13

Slide14

Slide15

Slide16

Control of the Cell Cycle

The Role of Protein

Kinases

Checkpoints

Kinase

Inhibitors

Cellular Responses

Slide17

DNA Replication

Semiconservative

Replication

According to this model of replication, each of the daughter duplexes should consist of one complete strand inherited from the parental duplex and one complete strand that has been newly synthesized

Replication of this type is said to be

semiconservative

because each daughter duplex contains one strand from the parent structure

Slide18

Slide19

Conservative replication

In

conservative replication,

the two original strands would remain together (after serving as templates), as would the two newly synthesized strands

As a result, one of the daughter duplexes would contain only parental DNA, while the other daughter duplex would contain only newly synthesized DNA.

Slide20

Slide21

Dispersive replication

In

dispersive replication

the parental strands are broken into fragments, and new strands are be synthesized in short segments

Then the old fragments and new segments are joined together to form a complete strand

As a result, the daughter duplexes contain strands that are composites of old and new DNA

Slide22

Slide23

DNA replication in bacteria

Replication begins at a specific site on the bacterial chromosome called the

origin

The origin of replication on the

E. coli

chromosome

is a specific sequence called

oriC

where a number of proteins bind to initiate the process of replication

Once initiated, replication proceeds outward from the origin in both directions, that is,

bidirectionally

The sites where the pair of replicated segments come together and join the

nonreplicated

DNA are termed

replication forks

Slide24

DNA replication in bacteria contd..

Each replication fork corresponds to a site where

The parental double helix is undergoing strand separation

Nucleotides are being incorporated into the newly synthesized complementary strands

The two replication forks move in opposite directions until they meet at a point across the circle from the origin, where replication is terminated

The two newly replicated duplexes detach from one another and are ultimately directed into two different cells

Slide25

Slide26

Unwinding the Duplex and Separating the Strands

Separation of the strands of a circular, helical DNA duplex poses major topological problems

Cells contain enzymes, called

topoisomerases

, that can change the state of

supercoiling

in a DNA

molecul

One enzyme of this type, called

DNA

gyrase

,

a type II

topoisomerase

, relieves the mechanical strain that builds up during replication in

E. coli

DNA

gyrase

molecules travel along the DNA ahead of the replication fork, removing positive

supercoils

Slide27

Slide28

Mechanism of action of DNA

gyrase

DNA

gyrase

removes positive

supercoils

by cleaving both strands of the DNA duplex, passing a segment of DNA through the double-stranded break to the other side, and then sealing the cuts

This process is driven by the energy released during ATP hydrolysis

Eukaryotic cells possess similar enzymes that carry out this required function.

Slide29

Properties of DNA Polymerases

DNA polymerases are the enzymes that synthesize new DNA strands

For the reaction to proceed, the enzyme requires the presence of DNA and all four

deoxyribonucleoside

triphosphates (

dTTP

,

dATP

,

dCTP

, and

dGTP

)

Original DNA strands serve as templates for the polymerization reaction

All DNA polymerases—both prokaryotic and eukaryotic—have the same two basic requirements

A template DNA strand to copy

A primer strand to which nucleotides can be added

Slide30

Semidiscontinuous

Replication

Both newly synthesized strands are assembled in a

5' → 3' direction

During the polymerization reaction, the —OH group at the 3' end of the primer carries out a

nucleophilic

attack on the 5'

α

-phosphate of the incoming nucleoside

triphosphate

The polymerase molecules responsible for construction of the two new strands of DNA both move in a 3'-to-5' direction along the template, and both construct a chain that grows from its 5'-P terminus

Slide31

Semidiscontinuous

Replication contd..

Consequently, one of the newly synthesized strands grows toward the replication fork, where the parental DNA strands are being separated, while the other strand grows away from the fork

The strand that grows away from the replication fork is synthesized

discontinuously

, as fragments

Before the synthesis of a fragment can be initiated, a suitable stretch of template must be exposed by movement of the replication fork

Once initiated, each fragment grows away from the replication fork toward the 5 end of a previously synthesized fragment to which it is subsequently linked.

Slide32

Semidiscontinuous

Replication contd..

The strand that is synthesized continuously is called the

leading strand

because its synthesis continues as the replication fork advances

The strand that is synthesized discontinuously is called the

lagging strand

because

initiation of each fragment must wait for the parental strands to separate and expose additional template

Because one strand is synthesized continuously and the other discontinuously, replication is said to be

semidiscontinuous

Slide33

Slide34

Okazaki fragments

The discovery that one strand was synthesized as small fragments was made by Reiji Okazaki of Nagoya University, Japan

Okazaki found that if bacteria were incubated in [3H]

thymidine

for a few seconds and immediately killed, most of the radioactivity could be found as part of small DNA fragments 1000 to 2000 nucleotides in length

In contrast, if cells were incubated in the labeled DNA precursor for a minute or two, most of the incorporated radioactivity became part of much larger DNA molecules

Slide35

Okazaki fragments

These results indicated that a portion of the DNA was constructed in small segments (later called

Okazaki fragments)

that were rapidly linked to longer pieces that had been synthesized previously

The enzyme that joins the Okazaki fragments into a continuous strand is called

DNA

ligase

Slide36

Role of

primase

enzyme

How does the synthesis of each of the Okazaki fragments begin, when none of the DNA polymerases are capable of strand initiation?

Further research studies revealed that initiation is not accomplished by a DNA polymerase but, rather, by a distinct type of RNA polymerase, called

primase

,

that constructs a short primer composed of RNA, not DNA

The leading strand, whose synthesis begins at the origin of replication, is also initiated by a

primase

molecule

Slide37

Role of

primase

enzyme contd..

The short RNAs synthesized by the

primase

at the 5' end of the leading strand and the 5' end of each Okazaki fragment serve as the required primer for the synthesis of DNA by a DNA polymerase

The RNA primers are subsequently removed, and the resulting gaps in the strand are filled with DNA and then sealed by DNA

ligase

Slide38

Slide39

Sequence of events at the replication fork

Replication involves more than incorporating nucleotides

Unwinding the duplex and separating the strands require the aid of two types of proteins that bind to the DNA

A

helicase

(or DNA unwinding enzyme)

Single-stranded DNA binding

(

SSB) proteins

Slide40

Role of DNA helicases

DNA

helicases

unwind a DNA duplex in a reaction that uses energy released by ATP hydrolysis to move along one of the DNA strands, breaking the hydrogen bonds that hold the two strands together and exposing the single-stranded DNA templates

E. coli

has at least 12 different

helicases

for use in various aspects of DNA (and RNA) metabolism

In bacteria, the

primase

enzyme and the

helicase

associate transiently to form what is called a “

primosome

”

Slide41

Role of SSB proteins

DNA unwinding by the

helicase

is aided by the attachment of SSB proteins to the separated DNA strands

These proteins bind selectively to single-stranded DNA, keeping it in an extended state and preventing it from becoming rewound or damaged

Slide42

Slide43

Slide44

Replication in Eukaryotic Cells

General features:

Eukaryotic cells replicate their genome in small portions, termed

replicons

Each

replicon

has its own origin from which replication forks proceed outward in both directions

In a human cell, replication begins at about 10,000 to 100,000 different replication origins

Approximately 10 to 15 percent of

replicons

are actively engaged in replication at any given time during the S phase of the cell cycle

Slide45

General features

contd

…

Replicons

located close together in a given chromosome tend to undergo replication simultaneously

The most highly compacted, least acetylated regions of the chromosome are packaged into heterochromatin, and they are the last regions to be replicated

The inactive, heterochromatic X chromosome in the cells of female mammals is replicated late in S phase, whereas the active,

euchromatic

X chromosome is replicated at an earlier stage

Slide46

The Eukaryotic Replication Fork

Slide47

Synthesis of the strands

The DNA of eukaryotic cells is synthesized in a

semidiscontinuous

manner

However, the Okazaki fragments of the lagging strand are considerably smaller than in bacteria, averaging about 150 nucleotides in length

Like DNA polymerase III of

E. coli,

the eukaryotic

replicative

DNA polymerase is present as a dimer

This suggests that the leading and lagging strands are synthesized in a coordinate manner by a single

replicative

complex, or

replisome

Slide48

Eukaryotic DNA polymerases

Five “classic” DNA polymerases have

been isolated from

eukaryotic cells, and they are designated α,

β

,

γ

,

δ

and

ε

Of these enzymes, polymerase

γ

replicates mitochondrial DNA, and polymerase

β

functions in DNA repair

The other three polymerases have

replicative

functions

Polymerase

α

is tightly associated with the

primase

, and together, they initiate the synthesis of each Okazaki fragment

Primase

initiates synthesis by assembly of a short RNA primer, which is then extended by the addition of about 20

deoxyribonucleotides

by polymerase

δ

Slide49

Eukaryotic DNA polymerases

contd

…

Polymerase

δ

is thought to be the primary DNA-synthesizing enzyme during replication of the lagging strand

Polymerase

ε

is thought to be the primary DNA-synthesizing enzyme during replication of the leading strand

After synthesizing an RNA-DNA primer, polymerase

α

is replaced at each template–primer junction by the PCNA–polymerase

δ

complex, which completes synthesis of the Okazaki fragment

Slide50

Eukaryotic DNA polymerases

contd

…

When polymerase

δ

reaches the 5' end of the previously synthesized Okazaki fragment, the polymerase continues along the lagging-strand template, displacing the primer

The displaced primer is cut from the newly synthesized DNA strand by an

endonuclease

(FEN-1) and the resulting nick in the DNA is sealed by a DNA

ligase

Like bacterial polymerases, all of the eukaryotic polymerases elongate DNA strands in the 5'→3' direction by the addition of nucleotides to a 3' hydroxyl group

None of them is able to initiate the synthesis of a DNA chain without a primer.

Slide51

Mutation

A mutation is any permanent, heritable change in the DNA base sequence of an organism

This altered DNA sequence can be reflected by changes in the base sequence of mRNA, and, sometimes, by changes in the amino acid sequence of a protein

Mutations can cause genetic diseases

They can also cause changes in enzyme activity, nutritional requirements, antibiotic susceptibility, morphology,

antigenicity

, and many other properties of cells

Slide52

Mutation

contd

…

A very common type of mutation is a single base alteration or point mutation

A

transition

is a point mutation that replaces a

purine

-pyrimidine base pair with a different

purine

-pyrimidine base pair

For example, an A-T base pair becomes a G-C base pair

A

transversion

is a point mutation that replaces a

purine

-pyrimidine base pair with a pyrimidine-

purine

base pair

For example, an A-T base pair becomes a T-A or a C-G base pair

Mutations are often classified according to the effect they have on the structure of the gene's protein product

This change in protein structure can be predicted using the genetic code table in conjunction with the base sequence of DNA or mRNA

Slide53

Mutation

contd

…

Slide54

Mutation

contd

…

Slide55

DNA REPAIR

Introduction

It is essential that the genetic information remain mostly unchanged as it is passed from cell to cell and individual to individual

DNA is one of the molecules in a cell that is most susceptible to environmental damage

When struck by ionizing radiation, the backbone of a DNA molecule is often broken; when exposed to a variety of reactive chemicals, many of which are produced by a cell’s own metabolism, the bases of a DNA molecule may be altered structurally

Slide56

Introduction

contd

…

When subjected to ultraviolet radiation, adjacent

pyrimidines

on a DNA strand have a tendency to interact with one another to form a covalent complex (called a dimer)

Even the absorption of thermal energy generated by metabolism is sufficient to split adenine and guanine bases from their attachment to the sugars of the DNA backbone

It is estimated that each cell of a warm-blooded mammal loses approximately 10,000 bases per day from these DNA damages

Slide57

Introduction contd

…

Failure to repair such DNA damages produces permanent alterations, or mutations, in the DNA

If the mutation occurs in a cell destined to become a gamete, the genetic alteration may be passed on to the next generation

Mutations also have effects in somatic cells in that they can interfere with transcription and replication, lead to the malignant transformation of a cell, or speed the process by which an organism ages

It is therefore essential that cells possess mechanisms for repairing DNA damage

Slide58

Importance of DNA repair

The importance of DNA repair can be appreciated by examining the effects on humans that result from DNA repair deficiencies;

Xeroderma pigmentosum

Cockayne

syndrome

Trichothiodystrophy

Bloom’s Syndrome

Fanconi’s

Anaemia

Hereditary

Nonpolyposis

Colon Cancer

Ataxia

telangiectasia

Humans beings possess enzymes that can directly repair damage from cancer-producing

alkylating

agents

Most repair systems, however, require that a damaged section of the DNA be

excised (

selectively removed)

Slide59

Repair systems

Nucleotide Excision Repair

This system operates by a cut-and-patch mechanism that removes a variety of bulky damages, including pyrimidine dimers and nucleotides to which various chemical groups have become attached

Two NER pathways exist, namely;

Transcription-coupled pathway

Global genomic pathway

Slide60

Nucleotide Excision Repair contd..

One of the key components of the NER repair machinery is TFIIH, a huge protein that also participates in the initiation of transcription

Two subunits of this protein (XPB and XPD) possess

helicase

activity

These enzymes separate the two strands of the duplex in preparation for removal of the damage

Slide61

Nucleotide Excision Repair contd..

The damaged strand is then cut on both sides of the lesion by a pair of

endonucleases

, and the segment of DNA between the incisions is released

Once excised, the gap is filled by a DNA polymerase, and the strand is sealed by DNA

ligase

Slide62

Slide63

Base Excision Repair

This repair system operates to remove altered nucleotides generated by reactive chemicals present in the diet or produced by metabolism

The process is initiated by a

DNA

glycosylase

that recognizes the alteration and removes the base by cleavage of the glycosidic bond holding the base to the deoxyribose sugar

Once an altered

purine

or pyrimidine is removed by a

glycosylase

, the “beheaded” deoxyribose phosphate remaining in the site is excised by the combined action of a specialized(AP)

endonuclease

and a DNA polymerase

Slide64

Base Excision Repair contd..

AP

endonuclease

cleaves the DNA backbone and a

phosphodiesterase

activity of polymerase

β

removes the sugar–phosphate remnant that had been attached to the excised base

Polymerase

β

then fills the gap by inserting a nucleotide complementary to the undamaged strand and the strand is sealed by DNA

ligase

III

Slide65

Mismatch Repair

A mismatched base pair causes a distortion in the geometry of the double helix that can be recognized by a repair enzyme

Thus, for a mismatch to be repaired after the DNA polymerase has moved past a site, it is important that the repair system distinguish the newly synthesized strand, which contains the incorrect nucleotide, from the parental strand, which contains the correct nucleotide

Slide66

Double-Strand Breakage Repair

X-rays, gamma rays, and particles released by radioactive atoms are all described as

ionizing radiation

because they generate ions as they pass through matter

Millions of gamma rays pass through our bodies every minute

When these forms of radiation collide with a fragile DNA molecule, they often break both strands of the double helix

Double-strand breaks (DSBs)

can

also be caused by certain chemicals, including the ones used in cancer chemotherapy (e.g.,

bleomycin

) , and free radicals produced by normal cellular metabolism

Slide67

Double-Strand Breakage Repair cntd

..

DSBs are also introduced during replication of damaged DNA

The predominant DSB repair pathway in mammalian cells is called

nonhomologous

end joining

In this pathway, a complex of proteins bind to the broken ends of the DNA duplex and catalyze a series of reactions that rejoin the broken strands

Another DSB repair pathway, known as

homologous recombination,

requires a homologous chromosome to serve as a template for repair of the broken strand.

Slide68

Slide69

Transcription

Slide70

Transcription

contd

…

Flow of information in a eukaryotic cell

The DNA of the chromosomes located within the nucleus contains the entire store of genetic information

Selected sites on the DNA are transcribed into pre-mRNAs (step 1), which are processed into messenger RNAs (step 2)

The messenger RNAs are transported out of the nucleus (step 3) into the cytoplasm, where they are translated into polypeptides by ribosomes that move along the mRNA (step 4)

After translation, the polypeptide folds to assume its native conformation (step 5)

Slide71

Transcription

contd

…

Transcription is a process in which a DNA strand provides the information for the synthesis of an RNA strand

The enzymes responsible for transcription in both prokaryotic and eukaryotic cells are called

DNA-dependent RNA polymerases,

or

simply

RNA polymerases

These enzymes are able to incorporate nucleotides, one at a time, into a strand of RNA whose sequence is complementary to one of the DNA strands, which serves as the

template

Slide72

Transcription

contd

…

The first step in the synthesis of an RNA is the association of the polymerase with the DNA template

The site on the DNA to which an RNA polymerase molecule binds prior to initiating transcription is called the

promoter

Cellular RNA polymerases are not capable of recognizing promoters on their own but require the help of additional proteins called

transcription factors

In addition to providing a binding site for the polymerase, the promoter contains the information that determines which of the two DNA strands is transcribed and the site at which transcription begins

Slide73

Transcription

contd

…

RNA polymerase moves along the template DNA strand toward its 5' end (i.e., in a 3'→5' direction)

As the polymerase progresses, the DNA is temporarily unwound, and the polymerase assembles a complementary strand of RNA that grows from its 5' terminus in a 3' direction

RNA polymerase catalyzes the reaction in which

ribonucleoside

triphosphate

substrates (NTPs) are cleaved into nucleoside monophosphates as they are polymerized into a covalent chain

Slide74

Reaction

catalysed

by RNA Polymerase

Slide75

Transcription

contd

…

As the polymerase moves along the DNA template, it incorporates complementary nucleotides into the growing RNA chain

A nucleotide is incorporated into the RNA strand if it is able to form a proper (Watson-Crick) base pair with the nucleotide in the DNA strand being transcribed

Once the polymerase has moved past a particular stretch of DNA, the DNA double helix reforms

Consequently, the RNA chain does not remain associated with its template as a DNA–RNA hybrid (except for about nine nucleotides just behind the site where the polymerase is operating)

Slide76

Uniqueness of RNA polymerases

RNA polymerases are capable of incorporating from about 20 to 50 nucleotides into a growing RNA molecule per second

Many genes in a cell are transcribed simultaneously by a hundred or more polymerases

Slide77

A look at the transcription bubble

Slide78

Transcription in Bacteria

Bacteria, such as

E. coli,

contain a single type of RNA polymerase composed of five subunits that are tightly associated to form a

core enzyme

Attachment of

sigma

(

σ

) factor to the core enzyme increases the enzyme’s affinity for promoter sites in DNA and decreases its affinity for DNA in general

As a result, the complete enzyme is thought to slide freely along the DNA until it recognizes and binds to a suitable promoter region

Slide79

Transcription in Bacteria

contd

…

The enzyme then separates (or

melts)

the two DNA strands in the region surrounding the start site

Strand separation makes the template strand accessible to the enzyme’s active site, which resides at the back wall of the channel

Once 10–12 nucleotides have been successfully incorporated into a growing transcript, the enzyme undergoes a major change in conformation and is transformed into a

transcriptional elongation complex

that can move

processively

along the DNA

Slide80

Bacterial promoters

Bacterial promoters are located in the region of a DNA strand just preceding the initiation site of RNA synthesis

The nucleotide at which transcription is initiated is denoted as +1 and the preceding nucleotide as -1

Those portions of the DNA preceding the initiation site (toward the 3' end of the template) are said to be

upstream

from that site

Those portions of the DNA succeeding it (toward the 5' end of the template) are said to be

downstream

from that site

Slide81

Bacterial promoters

contd

…

Analysis of the DNA sequences just upstream from a large number of bacterial genes reveals that two short stretches of DNA are similar from one gene to another

One of these stretches is centered at approximately 35 bases upstream from the initiation site and typically occurs as the sequence TTGACA

This TTGACA sequence (known as the -35 element) is called a

consensus sequence

Slide82

Bacterial promoters

contd

…

The second conserved sequence is found approximately 10 bases upstream from the initiation site and occurs at the consensus sequence TATAAT

This site in the promoter, named the

Pribnow

box

after its discoverer, is responsible for identifying the precise nucleotide at which transcription begins

Slide83

Features of bacterial promoters

Slide84

When/How is the process terminated?

Transcription in bacteria terminates when a specific nucleotide sequence is reached

In roughly half of the cases, a ring-shaped protein called

rho

is required for termination of bacterial transcription

Rho encircles the newly synthesized RNA and moves along the strand in a 3' direction to the polymerase, where it separates the RNA transcript from the DNA to which it is bound

In other cases, the polymerase stops transcription when it reaches a

terminator sequence

and releases the completed RNA chain without requiring additional factors

Slide85

Transcription in eukaryotic cells

Eukaryotic cells have three distinct transcribing enzymes in their cell nuclei

Each of these enzymes is responsible for synthesizing a different group of RNAs

Plants have two additional RNA polymerases that are not essential for life

A major distinction between transcription in prokaryotes and eukaryotes is the requirement in eukaryotes for a large variety of accessory proteins, or

transcription factors

Slide86

RNA polymerases in Eukaryotes

Slide87

Transcription in eukaryotic cells

contd

…

Transcription factors play a role in virtually every aspect of the transcription process, from the binding of the polymerase to the DNA template, to the initiation of transcription, to its elongation and termination

All three major types of eukaryotic RNAs—mRNAs,

rRNAs

, and

tRNAs

—are derived from precursor RNA molecules that are considerably longer than the final RNA product

The initial precursor RNA is equivalent in length to the full length of the DNA transcribed and is called the

primary transcript,

or

pre-RNA.

Slide88

Transcription in eukaryotic cells

contd

…

The corresponding segment of DNA from which a primary transcript is transcribed is called a

transcription unit

Slide89

Post-

transcripton

modifications/Processing

This is the process of converting the primary transcript into a mature messenger RNA

This conversion process entails;

Addition of a 5' cap

Addition of a 3' poly(A) tail to the ends of the transcript

Removal of any intervening

introns

Once processing is completed, the

mRNP

, which consists of mRNA and associated proteins, is ready for export from the nucleus

Slide90

5' capping

In the first step, the last of the three phosphates is removed, converting the 5 terminus to a

diphosphate

Then, a GMP is added in an

inverted

orientation so that the 5 end of the

guanosine

is facing the 5' end of the RNA chain

As a result, the first two nucleosides are joined by an unusual 5'–5‘

triphosphate

bridge

Finally, the terminal, inverted

guanosine

is

methylated

at the 7 position on its guanine base, while the nucleotide on the internal side of the

triphosphate

bridge is

methylated

at the 2' position of the ribose

The 5' end of the RNA now contains a

methylguanosine

cap

Slide91

Slide92

Polyadenylation

The 3 end of an mRNA contains a string of adenosine residues that forms a

poly(A) tail

The poly(A) tail invariably begins approximately 20 nucleotides downstream from the sequence AAUAAA

This sequence in the primary transcript serves as a recognition site for the assembly of a complex of proteins that carry out the processing reactions at the 3' end of the mRNA

The poly(A) processing complex is also physically associated with RNA polymerase II as it synthesizes the primary transcript

Slide93

Polyadenylation

contd

…

Among the proteins of the processing complex is an

endonuclease

that cleaves the

premRNA

downstream from the recognition site

Following cleavage by the nuclease, an enzyme called

poly(A) polymerase

adds 250 or so adenosines without the need of a template

The poly(A) tail together with an associated protein protects the mRNA from premature degradation by

exonucleases

Slide94

Slide95

RNA Splicing

Those parts of a primary transcript that correspond to the intervening DNA sequences (the

introns

) must be removed by a process known as

RNA splicing

To splice an RNA, breaks in the strand must be introduced at the 5' and 3' ends (the

splice sites)

of each

intron

, and the

exons

situated on either side of the splice sites must be covalently joined (

ligated

)

The splicing process occurs with absolute precision, because the addition or loss of a single nucleotide at any of the splice junctions would cause the resulting mRNA to be mistranslated

Slide96

Slide97

Importance of splicing

Changes in DNA sequence within a splice site can lead to the inclusion of an

intron

or the exclusion of an exon

It is estimated that approximately 15 percent of inherited human disease results directly from mutations that alter pre-mRNA splicing

In addition, much of the “normal” genetic variation in susceptibility to common diseases that is present in the human population may result from the effects of this variation on RNA splicing efficiency

Slide98

The genetic code

Introduction

The sequence of amino acids in a polypeptide is specified by the sequence of nucleotides in the DNA of a gene

The genetic code is the rules that specify how the nucleotide sequence of an mRNA is translated into the amino acid sequence of a polypeptide

The nucleotide sequence is read as triplets called

codons

The

codons

UAG, UGA

and

UAA

do not specify amino acids and are called

termination

codons

or

stop

codons

Slide99

Introduction

contd

…

Whenever one of these

codons

is encountered by a ribosome, it leads to termination of protein synthesis

AUG codes for

methionine

and also acts as an initiation (Start)

codon

Although

methionine

is found at internal positions in polypeptide chains, all eukaryotic polypeptides also start with

methionine

and all prokaryotic polypeptides start with a modified

methionine

(

N-

formyl

methionine

)

Slide100

Properties of the genetic code

1.

The genetic

codeis

degenerate

Since RNA is composed of four types of nucleotides, there are 64 possible

codons

This implies 64 possible triplets of nucleotides with different sequences

However, only 20 amino acids are commonly found in proteins

Most amino acids in proteins are specified by more than one

codon

(i.e. the genetic code is degenerate).

Codons

that specify the same amino acid (synonyms) often differ only in the third base

(the wobble position)

where base-pairing with the

anticodon

may be less stringent than for the first two positions of the codon

Slide101

Importance of degeneracy

As a result of the genetic code’s degeneracy, a mutation that changes only a single nucleotide in DNA (

point mutation),

and hence changes only a single nucleotide in the corresponding mRNA, often has no effect on the amino acid sequence of the encoded polypeptide

Slide102

Properties of the genetic code

contd

…

2.Universality of the genetic code

The genetic code is not universal but is the same in most organisms

Exceptions are found in mitochondrial genomes where some

codons

specify different amino acids to that normally encoded by nuclear genes

In mitochondria, the UGA

codon

does not specify termination of translation but instead encodes for tryptophan

Similarly, in certain protozoa UAA and UAG encode

glutamic

acid instead of acting as termination

codons

Slide103

Slide104

Translation

Introduction

A ribosome binds to an mRNA molecule and reads the nucleotide sequence from the 5 to 3 direction, synthesizing the corresponding protein from amino acids in an N-terminal (amino-terminal) to C-terminal (carboxyl terminal) direction

The amino acids used are covalently bound to

tRNA

(transfer RNA)molecules to form

aminoacyl

-

tRNAs

Each aminoacyl-tRNA bears a triplet of bases, called an

anticodon

Slide105

Translation

Introduction

contd

…

The ribosome reads each triplet

codon

of the mRNA in turn and an aminoacyl-tRNA molecule with an

anticodon

that is complementary to the

codon

binds to it via hydrogen bonding

A peptide bond is then formed between the incoming amino acid and the growing end of the polypeptide chain

Overall, protein synthesis

takes place in three stages

; initiation, elongation and termination

Slide106

Synthesis of aminoacyl-tRNA

During synthesis of the aminoacyl-tRNA, the amino acid is covalently bound to the A residue of the CCA sequence at the 3′ end

Each

tRNA

molecule carries only a single amino acid

However, because of the redundancy of the genetic code, several

codons

may encode the same amino acid and so there will also exist several types of

tRNA

with corresponding anticodons all bearing the same amino acid

The correct nomenclature is, for example,

tRNA

Gly

for the

tRNA

that will accept

glycine

whereas the corresponding aminoacyl-tRNA is

Gly-tRNA

Gly

Slide107

Slide108

Importance of

aminoacyl-tRNAs

synthesis

1. Each amino acid must be covalently linked to a

tRNA

molecule in order to take part in protein synthesis

This depends upon the ‘adaptor’ function of

tRNA

to ensure that the correct amino acids are incorporated

2. The covalent bond that is formed between the amino acid and the

tRNA

is a high energy bond that enables the amino acid to react with the end of the growing polypeptide chain to form a new peptide bond

For this reason, the synthesis of aminoacyl-tRNA is also referred to as

amino acid activation

Amino acids that

are not linked to

tRNAs

cannot be added to the growing polypeptide.

Slide109

Role of aminoacyl-tRNA synthetase

The attachment of an amino acid to a

tRNA

is catalyzed by an enzyme called

aminoacyl-tRNA synthetase

A separate aminoacyl-tRNA synthetase exists for every amino acid, making 20

synthetases

in total

The synthesis reaction occurs in two steps

The first step is the reaction of an amino acid and ATP to form an

aminoacyl-adenylate

(

also known as

aminoacyl

-AMP)

In the second step, without leaving the enzyme, the

aminoacyl

group of

aminoacyl

-AMP is transferred to the 3' end of the

tRNA

molecule to form aminoacyl-tRNA

Slide110

Initiation of protein synthesis

Each prokaryotic ribosome has three binding sites for

tRNAs

The

aminoacyltRNA

binding site (or A site)

is where, during elongation, the incoming aminoacyl-tRNA binds

The

peptidyl-

tRNA

binding site (or P site)

is where the

tRNA

linked to the growing polypeptide chain is bound

The

exit site (or E site)

is a binding site for

tRNA

following its role in translation and prior to its release from the ribosome

Slide111

Initiation of protein synthesis

contd

…

The first

codon

translated in all mRNAs is AUG which codes for

methionine

Naturally, other AUG

codons

also occur internally in an mRNA where they encode

methionine

residues internal to the protein

It is essential that the correct AUG is used as the initiation

codon

since this sets the correct reading frame for translation

A short sequence rich in

purines

(5-AGGAGGU-3), called the

Shine–

Dalgarno

sequence,

lies 5 to the AUG initiation

codon

Slide112

Initiation of protein synthesis

contd

…

This sequence is complementary to part of the 16S

rRNA

in the small ribosomal subunit

Therefore this is the binding site for the 30S ribosomal subunit which then migrates in a 3 direction along the mRNA until it encounters the AUG initiation

codon

Thus the Shine–

Dalgarno

sequence delivers the ribosomal subunit to the correct AUG for initiation for translation

Initiation of protein synthesis is catalyzed by proteins called

initiation factors

(

IFs)

In prokaryotes, three initiation factors (IF1, IF2 and IF3) are essential

Slide113

Slide114

Elongation

At the start of the first round of elongation

,

the initiation

codon

(AUG) is positioned in the P site with

fMet-tRNA

f

Met

bound to it via

codon–anticodon

base-pairing

The next

codon

in the mRNA is positioned in the A site

Elongation of the polypeptide chain occurs in three steps called the

elongation cycle,

namely aminoacyl-tRNA binding, peptide bond formation and translocation

Slide115

Slide116

Termination

After the elongation, eventually, one of three termination

codons

(also called Stop

codons

) becomes positioned in the A site

Unlike other

codons

, prokaryotic cells do not contain

aminoacyl-tRNAs

complementary to stop

codons

Instead, one of two

release factors (RF1 and RF2)

binds instead

RF1 recognizes UAA and UAG whereas RF2 recognizes UGA

A third release factor,

RF3,

is also needed to assist RF1 or RF2

Thus either RF1+RF3 or RF2+RF3 bind depending on the exact termination

codon

in the A site

Slide117

Termination

contd

…

RF1 (or RF2) binds at or near the A site whereas RF3/GTP binds elsewhere on the ribosome.

The release factors cause the peptidyl transferase to transfer the polypeptide to a water molecule instead of to aminoacyl-tRNA, effectively cleaving the bond between the polypeptide and

tRNA

in the P site

The polypeptide, now leaves the ribosome, followed by the mRNA and free

tRNA

, and the ribosome dissociates into 30S and 50S subunits ready to start translation afresh

Slide118

Translation in Eukaryotes

How does the process of translation take place in eukaryotes?

Slide119

Slide120

REGULATION OF GENE EXPRESSION

In Bacteria:

A bacterial cell lives in direct contact with its environment, which may change dramatically in chemical composition from one moment to the next

At certain times, a particular compound may be present, while at other times that compound is absent

Slide121

The bacterial

operon

In bacteria, the genes that encode the enzymes of a metabolic pathway are usually clustered together on the chromosome in a functional complex called an

operon

A typical bacterial

operon

consists of;

Structural genes

A promoter region

An operator region

A regulatory gene

Slide122

Structural Genes

These are the genes that code for the enzymes

The structural genes of an

operon

usually lie adjacent to one another

The RNA polymerase moves from one structural gene to the next, transcribing all of the genes into a single mRNA

This extended mRNA is then translated into the various individual enzymes of the metabolic pathway

Consequently, turning on one gene turns on all the enzyme-producing genes of an

operon

Slide123

The promoter

It is the site where the RNA polymerase binds to the DNA prior to beginning transcription

The regulatory gene

This gene encodes the repressor protein

Slide124

The operator

The operator typically resides adjacent to or overlaps with the promoter

It serves as the binding site for a protein, called the

repressor

The repressor is an example of a

gene regulatory protein

—a protein that recognizes a specific sequence of base pairs within the DNA and binds to that sequence with high affinity

DNA-binding proteins, such as bacterial repressors, play a predominant role in determining whether or not a particular segment of the genome is transcribed

Slide125

Diagrammatic representation of an

operon

Slide126

What determines

operon

expression?

The key to

operon

expression lies in the repressor

When the repressor binds the operator, the promoter is shielded from the polymerase, and transcription of the structural genes is prevented

The capability of the repressor to bind the operator and inhibit transcription depends on the conformation of the repressor, which is regulated

allosterically

by a key compound in the metabolic pathway, such as lactose or tryptophan

It is the concentration of this key metabolic substance that determines whether the

operon

is active or inactive at any given time

Slide127

The

lac

Operon

It is a cluster of genes that regulates production of the enzymes needed to degrade lactose in bacterial cells

The

lac

operon

is an example of an

inducible

operon

,

i.e

one in which the presence of a key metabolic substance (in this case, lactose) induces transcription of the structural genes

The

lac

operon

contains three tandem structural genes, namely;

z

gene

y

gene

a

gene

Slide128

The

lac

Operon

contd

….

The

z gene

encodes

β

-

galactosidase

The

y gene

encodes

galactoside

permease

, a protein that promotes the entry of lactose into the cell

The

a gene

encodes

thiogalactoside

transacetylase

, an enzyme whose physiologic role is unclear

If lactose is present in the medium, the disaccharide enters the cell where it binds to the

lac

repressor, changing the conformation of the repressor and making it unable to attach to the DNA of the operator

In this state, the structural genes are transcribed, the enzymes are synthesized, and the lactose molecules are catabolized

Slide129

The

lac

Operon

contd

….

In an inducible

operon

, such as the

lac

operon

,

the repressor protein is able to bind to the DNA only in the absence of lactose, which functions as the

inducer

As the concentration of lactose in the medium decreases, the disaccharide dissociates from its binding site on the repressor molecule

Release of lactose enables the repressor to bind to the operator, which physically blocks the polymerase from reaching the structural genes, turning off transcription of the

operon

Slide130

How does cAMP

influence the expression of the

lac

operon

?

Slide131

The trp

Operon

In a

repressible

operon

, such as the tryptophan (or

trp

)

operon

, the repressor is unable to bind to the operator DNA by itself

Instead, the repressor is active as a DNA-binding protein only when complexed with a specific factor, such as tryptophan which functions as a

corepressor

In the absence of tryptophan, the operator site is open to binding by RNA polymerase, which transcribes the structural genes of the

trp

operon

and leads to the production of the enzymes that synthesize tryptophan

Once tryptophan becomes available, the enzymes of the tryptophan synthetic pathway are no longer required

Slide132

The

trp

Operon

contd

….

Under these conditions, the increased concentration of tryptophan leads to the formation of the tryptophan–repressor complex, which blocks transcription

Thus, when tryptophan concentration is high, the

operon

is repressed, preventing overproduction of tryptophan

When the tryptophan concentration is low, most repressor molecules lack a

corepressor

and therefore fail to attach to the operator

Genes are transcribed, enzymes are synthesized, and the needed end-product (tryptophan) is manufactured

Slide133

Slide134

Slide135

Regulation of gene expression in eukaryotes

Regulation of gene expression in eukaryotic cells occurs primarily at three distinct levels, namely;

Transcriptional-level control

mechanisms determine whether a particular gene can be transcribed and, if so, how often

Processing-level control

mechanisms determine the path by which the primary mRNA transcript (pre-mRNA) is processed into a messenger RNA that can be translated into a polypeptide

Translational-level control

mechanisms determine whether a particular mRNA is actually translated and, if so, how often and for how long a period

Slide136

Slide137

Polymerase Chain Reaction

In 1983, a new technique was conceived by

Kary

Mullis of

Cetus

Corporation that has become widely used to amplify specific DNA fragments

PCR amplification is readily adapted to RNA templates by first converting them to complementary DNAs using reverse transcriptase

The technique employs a heat-stable DNA polymerase, called

Taq

polymerase,

that was originally isolated from

Thermus

aquaticus

,

a bacterium that lives in hot springs at temperatures above 90°C

Slide138

Steps in PCR

In the simplest protocol, a sample of DNA is mixed with an aliquot of

Taq

polymerase

and all four

deoxyribonucleotides

, along with a large excess of two short, synthetic DNA fragments (

oligonucleotides

) that are complementary to DNA sequences at the 3' ends of the region of the DNA to be amplified

The

oligonucleotides

serve as primers to which nucleotides are added during the following replication steps

The mixture is then heated to about 95°C, which is hot enough to cause the DNA molecules in the sample to separate into their two component strands

Slide139

Steps in PCR

contd

…

The mixture is then cooled to about 60°C to allow the primers to hybridize to the strands of the target DNA, and the temperature is raised to about 72°C, to allow the

thermophilic

polymerase to add nucleotides to the 3' end of the primers

As the polymerase extends the primer, it selectively copies the target DNA, forming new complementary DNA strands

The temperature is raised once again, causing the newly formed and the original DNA strands to separate from each other

Slide140

Steps in PCR

contd

…

The sample is then cooled to allow the synthetic primers in the mixture to bind once again to the target DNA, which is now present at twice the original amount

This cycle is repeated over and over again, each time doubling the amount of the specific region of DNA that is flanked by the bound primers

Billions of copies of this one specific region can be generated in just a few hours using a

thermal cycler

that automatically changes the temperature of the reaction mixture to allow each step in the cycle to take place

Slide141

Slide142

Applications of PCR

1. Amplifying DNA for Cloning or Analysis

This is particularly helpful in cases where the source DNA is very scarce, since PCR can generate large amounts of DNA from minuscule samples, such as that in a single cell

PCR has been used in criminal investigations to generate quantities of DNA from a spot of dried blood left on a crime suspect’s clothing or even from the DNA present in part of a single hair follicle left at the scene of a crime

For this purpose, one selects regions of the genome for amplification that are highly polymorphic (i.e., vary at high frequency within the population), so that no two individuals will have the same-sized DNA fragments

Slide143

Applications of PCR

contd

…

2. Testing for the Presence of Specific DNA Sequences

This is particularly useful when one wants to determine whether or not a tissue sample contains a particular virus

In such a

scenario,nucleic

acid is isolated from the sample and PCR primers complementary to the viral DNA are added, along with the other PCR reagents. The reaction is then allowed to proceed

If the virus genome is present in the sample, the PCR primers will hybridize to it and the PCR reaction will generate a product

Slide144

Applications of PCR contd…

If the virus is not present, the PCR primers will not hybridize and no product will be generated

Thus, in this application, the PCR reaction itself serves as the detection system.

Slide145

Applications of PCR contd

…

3.Comparing DNA Molecules

If two DNA molecules have the same base sequence, they will yield the same PCR products in reactions with identical primers

This is the premise for quick assays that compare the similarity of two DNA samples such as genomic DNA from bacterial isolates

PCR is performed on the samples using several primers, which can be specifically designed or randomly generated

The products are separated by gel electrophoresis and compared

The more similar the sequences of the bacterial genomes, the more similar their PCR products will be.

Slide146

Applications of PCR

contd

…

4. Quantifying DNA or RNA Templates

PCR can also be used to determine how much of a specific nucleotide sequence (DNA or RNA) is present in a mixed sample

One approach to this quantitative PCR uses the binding of a dye specific for double-stranded DNA to quantify the amount of double-stranded product being generated

The rate of accumulation of product is proportional to the amount of template present in the sample

Slide147

GENE THERAPY

Gene therapy is the genetic alteration of cells to correct the effects of a disease-causing mutation

In humans, gene therapy is performed on somatic cells (somatic cell gene therapy)

Because only somatic cells are affected, the alteration is not transmitted to offspring

Another form,

germline

gene therapy, affects all cells of the body, resulting in transmission of the alteration to offspring

This form of gene therapy is not currently practiced with humans

Slide148

Somatic cell gene therapy

In somatic cell gene therapy, a DNA sequence is inserted into a somatic cell to correct a mutation

Cells may be removed from the patient for manipulation and subsequent reinsertion

(ex vivo

therapy), or they may be manipulated without removal from the patient

(in vivo

therapy)

Ideally, cells with a very long life span (e.g., bone marrow stem cells) are treated, but other cells (e.g., lymphocytes) are sometimes more practical targets

Slide149

Gene Replacement Therapy

Most gene therapy protocols currently under way involve the replacement of a missing gene product in a cell (e.g., the replacement of clotting factor VIII in a hemophilia A patient)

Recombinant DNA techniques are used to insert a normal DNA sequence into a vector, which then carries the DNA into the patient's cells, where it supplies a template for the normal gene product

Slide150

Vectors used to deliver genes into cells

Retroviruses

Adenoviruses

Adeno

-Associated viruses

Liposomes

Slide151

Use of Recombinant DNA Techniques in Medicine

Knowledge of the following tools and processes will be of great help in order to understand the applications:

Restriction enzymes

Cloning vectors

Polymerase

chain

reaction

Gel electrophoresis

Nucleic

acid

hybridization

Expression vectors

Slide152

Restriction Enzymes

Slide153

Restriction

Enzymes

Slide154

Cloning Vectors

Slide155

Gel Electrophoresis

Slide156

Nucleic

A

cid

H

ybridization

Slide157

rDNA

techniques

f

or prevention & treatment of disease

Vaccines e.g. hepatitis

B

virus

Production of Therapeutic

Proteins

e.g

:

Insulin

And Growth

Hormone

Complex Human Proteins like Hematopoietic growth factors, Tissue

plasminogen

activator (TPA), factor

VIII

Genetic Counseling

Gene Therapy

Slide158

r

DNA

techniques for diagnosis of disease

DNA Polymorphisms-due to point mutations,

deletions

and insertions

DNA

Polymorphisms can be detected by:

Restriction

Fragment Length

Polymorphisms

Allele-Specific

Oligonucleotide

Probes

PCR

Repetitive DNA e.g. variable number of tandem repeats (VNTR

)

DNA

Chips