chapter 6 : expression of biological information...ed the synthesis of short rna primer (using dna...

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BIOLOGY NOTES

CHAPTER 6 : EXPRESSION OF BIOLOGICAL INFORMATION

SUBTOPIC 6.1: DNA and genetic information

LEARNING OUTCOMES: a) State the concept of Central Dogma

MAIN IDEAS

/KEY POINT EXPLANATION NOTES

Central Dogma

Definition: The ‘Central Dogma’ is the process by which the

instructions in DNA are converted into a functional product. It

was first proposed in 1958 by Francis Crick, discoverer of the

structure of DNA.

The central dogma of molecular biology explains the

flow of genetic information, from DNA to RNA to make

a functional product, a protein.

The central dogma suggests that DNA contains the

information needed to make all of our proteins, and that

RNA is a messenger that carries this information to

the ribosomes.

The ribosomes serve as factories in the cell where the

information is ‘translated’ from a code into the

functional product.

The process by which the DNA instructions are

converted into the functional product is called gene

expression.

Gene expression has two key stages- transcription and

translation.

In transcription, the information in the DNA of every

cell is converted into small, portable RNA messages.

During translation, these messages travel from where the

DNA is in the cell nucleus to the ribosomes where they

are ‘read’ to make specific protein.

The central dogma states that the pattern of information

that occurs most frequently in our cells is:

o From existing DNA to make new DNA (DNA

replication)

o From DNA to make new RNA (transcription)

o From RNA to make new proteins (translation)

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Concept of Central Dogma: An illustration showing the flow of information between DNA, RNA

and protein.

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BIOLOGY NOTES


SUBTOPIC 6.2 : DNA Replication

LEARNING OUTCOMES: a) Explain the semi-conservative replication of DNA.

b) Explain the enzymes and proteins involved in DNA replication.

c) Explain the mechanism of DNA replication and the enzymes involved.

MAIN IDEAS


Models of

DNA

replication:

Semi-

conservative

DNA replication is the biological process of producing two

identical replicas of DNA from one original DNA molecule.

DNA is made up of a double helix of two complementary

strands.

During replication, these strands are separated. Each strand

of the original DNA molecule then serves as a template for

the production of its counterpart, a process referred to

as semi-conservative replication.

This semi-conservative replication model has been

demonstrated by Meselson and Stahl.

As a result of semi-conservative replication, the new helix

will be composed of an original DNA strand as well as a

newly synthesize strand.

Cellular proofreading and error-checking mechanisms ensure

near perfect fidelity for DNA replication

Enzymes and

proteins

involved in

DNA

replication

No. Enzyme/Protein Function

1 Helicases

catalyzed the untwist the double

helix at the replication forks,

separating the two parental strands

and making them available as

template strands.

https://en.wikipedia.org/wiki/DNA

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MAIN IDEAS


2 Topoisomerase

The untwisting of the double helix

causes tighter twisting and strain

ahead of the replication fork.

Topoisomerase catalyzed in

relieving this strain by breaking,

swiveling and rejoining DNA

strands.

3 Primase

Catalysed the synthesis of short

RNA primer (using DNA strand as

a template), generally 5-10

nucleotides long.

4 DNA

polymerase I

Cataysed the removal of primer

from the 5’end and replacing it

with DNA nucleotides added one

by one to the 3’ end.

5 DNA

polymerase III

Cataysed the synthesizing new

DNA strand by adding nucleotides

to an RNA primer or a pre-

existing DNA strand by using

parental DNA strands as a

template.

6 DNA ligase

Cataysed the joining of Okazaki

fragments of lagging strand; on

leading strand, catalyzed the

joining of 3’ end of DNA that

replaces primer to rest of leading

strand DNA.

7 Single-strand

binding proteins

Bind to the unpaired DNA strands,

keeping them from re-pairing/

stabilized the unwound parental

strands.

• Special properties of DNA polymerase III:

• Cannot initiate the synthesis of a DNA strand all by itself.

• Need an RNA primer

• Complementary to the parental DNA strand

• Can only add new nucleotides in 5’ to 3’ end direction.

• Can only add a nucleotide to the 3' end of an already

growing chain

• Has important implications for antiparallel strands

running in opposite directions.

Mechanism of

DNA

replication and

the enzymes

involved

Step 1: Replication Fork Formation

• Before DNA can be replicated, the double stranded molecule

must be “unzipped” into two single strands.

• In order to unwind DNA, these interactions between base pairs

must be broken. This is performed by an enzyme known as DNA

helicase.

• DNA helicase disrupts the hydrogen bonding between base pairs

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MAIN IDEAS


to separate the strands into a Y shape known as the replication

fork. This area will be the template for replication to begin.

• The replication fork is bi-directional; one strand is oriented in the

3' to 5' direction (leading strand) while the other is oriented 5' to

3' (lagging strand).

• After the parental strands separate, single-strand binding

proteins bind to the unpaired DNA strands, keeping them from

re-pairing.

• The untwisting of the double helix causes tighter twisting and

strain ahead of the replication fork. Topoisomerase helps relieve

this strain by breaking, swiveling, and rejoining DNA strands.

Step 2: Primer Binding

• Once the DNA strands have been separated, a short piece of RNA

called a primer binds to the 3' end of the strand.

• The primer always binds as the starting point for replication.

Primers are generated by the enzyme primase.

• Primase starts a complementary RNA chain from single RNA

nucleotide, adding more RNA nucleotides one at a time, using

DNA strand as a template.

• The completed primer, generally 5-10 nucleotides long.

• The new DNA strand will start from 3’ end of the RNA primer.

Step 3: Elongation

• Enzymes known as DNA polymerases are responsible creating

the new strand by a process called elongation.

• DNA polymerase III binds to the strand at the site of the primer

and begins adding new base pairs complementary to the RNA

primer and then continues adding DNA nucleotides,

complementary to the parental DNA strand template strand, to the

growing end of the new DNA strand.

• Because replication proceeds in the 5' to 3' direction on the

leading strand, the newly formed strand is continuous.

• The lagging strand begins replication by binding with multiple

primers. Each primer is only several bases apart.

• DNA polymerase III then adds pieces of DNA, called Okazaki

fragments, to the strand between primers.

• This process of replication is discontinuous as the newly created

fragments are disjointed.

• After Okazaki fragment forms, DNA polymerase I, replaces the

RNA nucleotides of the adjacent primer with DNA nucleotides.

Step 4: Termination

• Once both the continuous and discontinuous strands are formed,

an enzyme called exonuclease removes all RNA primers from the

original strands.

• These primers are then replaced with appropriate bases.

Another exonuclease “proofreads” the newly formed DNA to

check, remove and replace any errors.

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MAIN IDEAS


• Another enzyme called DNA ligase joins Okazaki fragments

together forming a single unified strand.

• The ends of the linear DNA present a problem as DNA

polymerase can only add nucleotides in the 5′ to 3′ direction.

• The ends of the parent strands consist of repeated DNA sequences

called telomeres. Telomeres act as protective caps at the end of

chromosomes to prevent nearby chromosomes from fusing.

• A special type of DNA polymerase enzyme called telomerase

catalyzes the synthesis of telomere sequences at the ends of the

DNA.

• Once completed, the parent strand and its complementary DNA

strand coils into the familiar double helix shape. In the end,

replication produces two DNA molecules, each with one strand

from the parent molecule and one new strand.

• Differences between leading strand and lagging strand.

Leading strand Lagging strand

Synthesized continuously

TOWARDS replication fork

Synthesized discontinuously

AWAY from replication fork

No formation of Okazaki

fragments

Formation of several Okazaki

fragments

BOTH require RNA primer to initiate replication

Mechanism of DNA replication and the enzyme involved.

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BIOLOGY NOTES


SUBTOPIC 6.3: Protein synthesis : Transcription and translation

LEARNING OUTCOMES: a) Explain briefly transcription and translation

b) Introduce codon and its relationship with sequence of amino acid using genetic

code table.

c) Explain transcription and the stage involved (initiation, elongation and

termination) in the formation of mRNA strand from 5’ to 3’.

d) Explain translation and the stages involved in translation:

i. initiation

ii. elongation (codon recognition, peptide bond formation and translocation)

iii. termination

MAIN IDEAS


Roles of

transcription

and translation

Definition of protein synthesis:

the process by which amino acids are linearly arranged into proteins

through the involvement of ribosomal RNA, transfer RNA, messenger

RNA, and various enzymes.

Transcription:

Transcription is the first step of gene expression, in which a particular

segment of DNA is copied into RNA by the enzyme RNA polymerase.

Both DNA and RNA are nucleic acids, which use base pairs of

nucleotides as a complementary language.

Translation:

In translation, messenger RNA (mRNA) is decoded in a ribosome,

outside the nucleus, to produce a specific amino acid chain, or

polypeptide. The polypeptide later folds into an active protein and

performs its functions in the cell.

Importance of

Protein

Synthesis

There are many different types of proteins and associated functions.

Some more commonly used examples are:

Enzymes are protein molecules that catalyze biochemical reactions.

Common examples are enzymes involved in digestion. Amylase,

which breaks down starch into sugars and is present in the saliva of

mammals. Pepsin and trypsin are enzymes involved in the protein

digestion. Pepsin and trypsin cleaves the large protein molecules into

shorter polypeptides which can be passed through the lining of the

small intestine.

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MAIN IDEAS


Hormones are proteins that are able to transmit signals from one body

location to another. Insulin is an extracellular protein and regulates the

metabolism of glucose controlling the levels of blood sugar.

Contractile proteins, like actin and myosin in the muscles, are

involved in movement.

Structural protein is usually filamentous and are used to provide

support. Keratin strengthen protective coverings such as hair and

nails. Collagen and elastin are important component of the connective

tissue, which build tendons and ligaments.

Transport proteins supply different cellular processes with the

required ions, small molecules, or macromolecules, such as another

protein. Most common transport proteins are integral membrane

proteins they are involved in the transport across a biological

membrane.

Antibodies are another class of protein which are involved in immune

response. Their primary function is to bind to foreign for the body

substances and thus to identify them for destruction. Antibodies are

usually anchored in the membranes of the immune response cells or are

excreted into the extracellular matrix.

Transcription

In transcription, a portion of the double-stranded DNA template gives

rise to a single-stranded RNA molecule. This process occurs in

nucleus as it copies DNA into mRNA.

Enzyme involved in this process is RNA polymerase.

Functions:

1. Select which gene to transcribe

2. Recognizes which of the 2 paired DNA strands it should copy

(act as template)

3. Identifies where it should begin and end transcription

4. Unwinds DNA double helix by breaking the hydrogen bonds

5. Adding new RNA nucleotides in 5’ – 3’ direction, doesn’t

requires a primer

6. Rewinds the DNA strands

Stages : 1. Initiation

2. Elongation

3. Termination

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MAIN IDEAS


1. Initiaition:

The first step in transcription is initiation, when the RNA

polymerase binds to the DNA upstream (5′) of the gene at a

specialized sequence called a promoter.

The promoter of a gene includes within in the transcription start

point - the nucleotide where RNA polymerase actually begins

synthesis of the mRNA – and typically extends several dozen or

so nucleotide pair upstream from the start point.

RNA polymerase binds in a precise location and orientation on the

promoter. This in turn determines where transcription starts and

which of the two strands of DNA helix is used as the template.

In eukaryotes, a collection of protein called transcription factors

mediate the binding of RNA polymerase and the initiation of

transcription (Only after transcription factors are attached to the

promoter does RNA polymerase bind to it).

Once the appropriate transcription factors are firmly attached to

the promoter DNA and the RNA polymerase is bound to them in

the correct orientation on DNA, the enzyme unwinds the two

DNA strands and begins transcribing the template strand at the

start point.

2. Elongation:

As RNA polymerase moves along DNA, it untwists the double

helix, exposing about 10-20 DNA nucleotides at a time for pairing

with RNA nucleotides.

The enzyme adds nucleotides to the 3’ end of the growing RNA

molecule as it continues along the double helix.

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MAIN IDEAS


Transcription progress at a rate of about 40 nucleotides per second

in eukaryotes.

A single gene can be transcribed simultaneously by several

molecules of RNA polymerase. The congregation of many

polymerase molecules simultaneously transcribing a single gene

increases the amount of mRNA transcribed from it, which helps

the cell make the encoded protein in large amounts.

3. Termination:

Bacteria and eukaryotes differ in the way they terminate

transcription.

In bacteria, transcription proceeds through a terminator

sequence in the DNA. The transcribed terminator functions as

the terminator signal, causing the polymerase to detach from

the DNA and release the transcript (requires no further

modification before translation).

In eukaryotes, RNA polymerase transcribed a sequence on the

DNA called polyadenylation signal sequence, which specifies

a signal to cut the RNA transcript free from RNA polymerase,

releasing the pre-mRNA.

The pre-mRNA then undergoes splicing process. During this

process, certain interior sections of RNA molecule / non-

coding region (intron) are cut out and the remaining parts/

coding region (exon) spliced together producing an mRNA

molecule that ready for translation.

How is pre-mRNA spicing carried out?

- The removal of introns is accomplished by a large

complex made of protein and small RNAs called

spliceosome.

- This complex binds to several short nucleotide

sequence along an intron, the intron is then release

(and rapidly degraded), and spliceosome joins together

the two exons that flanked the intron.

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MAIN IDEAS


Relationship

between base

sequences in

codons with

specific amino

acids using

genetic code

table

Codon:

A triplets of nucleotides within mRNA that codes

for an amino acid. Codon are customarily written in the 5’ 3’

direction.

Characteristics:

• 1 codon consists of triplet nucleotide combination.

o There are 64 codons in genetic code table

o Only 61 codon specify amino acids

• One start codon, AUG o Encodes for Methionine (Met)

o Since AUG is the start codon and codes for methionine,

do all protein have methionine as the first amino acid?

Explain.

• 3 stop codons: UAA,UGA, UAG o Termination codons

o Do not specify amino acids.

• Non-overlapping - E.g : Codon 5’ AUGUCUAGU 3’ read as 5’ AUG, UCU,

AGU 3’ NOT 5’ AUG, GUC, CUA, AGU 3’

• 1 codon specify for particular amino acid

• 1 amino acid encoded by several (1/more) codon(s)

Translation

• During translation, the sequence of codons along an mRNA

molecule is decoded, or translated, into a sequence of amino acids

making up a polypeptide chain.

• This process occurs in cytoplasm.

• Fascilitates by transfer RNA/ tRNA.

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MAIN IDEAS


• Anticodon:

A nucleotide triplet at one end of a tRNA molecule that base pairs

with a particular complementary to codon on an mRNA molecule.

Anticodons are conventionally written 3’ 5’ to align properly

with codons written 5’ 3’.

• Before translation can takes place:

– Each amino acid is matched with the correct tRNA by a

specific enzyme called aminoacyl-tRNA synthetases.

– This process is known as: activation of amino acids.

Stages of translation:

1. Initiation:

The start codon (AUG) signals the start of translation (this is

important because it establishes the codon reading frame for the

mRNA)

In the first step of translation, small ribosomal subunit binds to

both the mRNA and specific initiator tRNA, which carries the

amino acid methionine.

In eukaryotes, the small subunit with the initiator tRNA

already bound, binds to the 5’ cap of the mRNA and then moves

(or scans), downstream along mRNA until it reaches the start

codon; the initiator tRNA then hydrogen-bonds to the AUG start

codon.

The first components to associate with each other during the

initiation stage of translation are mRNA, a tRNA bearing the

first amino acid of the polypeptide, and the small ribosomal

subunit.

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MAIN IDEAS


This followed by the attachment of a large ribosomal subunit,

completing the translation initiation complex.

At the completion of the initiation process, the initiator tRNA

sits in the P site of the ribosome, and the vacant A site is ready

for the next aminoacyl tRNA.

2. Elongation:

In the elongation stage of translation, amino acids are added

one by one to the previous amino acids at the C-terminus of

growing chain.

The mRNA is moved through the ribosome in one direction

only, 5’ end first; this is equivalent to the ribosome moving 5’

3’ on the mRNA

Involve 3 process; codon recognition, peptide bond formation

and translocation.

During codon recognition: Anticodon of the 2nd aminoacyl-

tRNA base-pairs with the complementary 2nd codon in A site

Peptide bond formation: A peptide bond between the two

amino acids is formed. The reaction is catalyzed by rRNA

molecule of large ribosomal subunit

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MAIN IDEAS


Polypeptide from initiator tRNA in P site is removed and

attaches to the 2nd tRNA in the A site

During translocation: Ribosome moves along mRNA,

translocating the 2nd tRNA in the A site to P site

The initiator-tRNA is released and A site is occupied by the 3rd

aminoacyl-tRNA

The whole mechanism repeats several times until all the codes

has been translated

3. Termination:

The final stage of translation is termination where the

elongation continues until a stop codon in mRNA reaches the

A site.

The nucleotide base triplets UAG, UAA and UGA do not code

for amino acids but instead act as signals to stop translation.

A release factor (a protein shaped like an aminoacyl tRNA)

binds directly to the stop codon in the A site.

The release factor causes the addition of a water molecule

instead of an amino acid to polypeptide chain (water molecule

are abundant in the cytosol).

This reaction hydrolysed the bond between the completed

polypeptide abd the tRNA in the P site, releasing the

polypeptide through the exit tunnel of the ribosome’s large

subunit.

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The stages involved in translation.

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BIOLOGY NOTES


SUBTOPIC 6.4: Gene regulation and expression – lac operon

LEARNING OUTCOMES: a) Explain the concept of operon and gene regulation.

b) State the components of operon.

c) Explain the components of lac operon and their functions in E.coli

c) Explain the mechanism of the operon in the absence and presence of

lactose.

MAIN IDEAS


Concept of

operon and gene

regulation

• Operon is a genetic regulatory system found in bacteria

and their viruses in which genes coding for functionally

related proteins are clustered along the DNA.

• This feature allows protein synthesis to be controlled

coordinately in response to the needs of the cell.

• By providing the means to produce proteins only when and

where they are required, the operon allows the cell to

conserve energy (which is an important part of an

organism’s life strategy)

Components of

operon

• A typical operon consists of a group of structural genes that

code for enzymes involved in a metabolic pathway, such as

the biosynthesis of an amino acid.

• These genes are located contiguously on a stretch of DNA

and are under the control of one promoter (a short segment

of DNA to which the RNA polymerase binds to initiate

transcription).

• A single unit of messenger RNA (mRNA) is transcribed

from the operon and is subsequently translated into separate

proteins.

• The promoter is controlled by various regulatory elements

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that respond to environmental cues.

• One common method of regulation is carried out by a

regulator protein that binds to the operator region, which

is another short segment of DNA found between the

promoter and the structural genes.

• The regulator protein can either block transcription, in

which case it is referred to as a repressor protein; or as an

activator protein it can stimulate transcription.

• Further regulation occurs in some operons: a molecule

called an inducer can bind to the repressor, inactivating it;

or a repressor may not be able to bind to the operator unless

it is bound to another molecule, the corepressor.

Components of

lac operon and

their functions

in E.coli

• Lac operon: a system that allows the Escherichia coli to

repress the production of enzymes involved in lactose

metabolism when lactose is present and glucose is absent.

• Regulator that turn the operon "on" and "off" in response to

lactose and glucose levels called repressor protein.

• The repressor protein acts as a lactose sensor. It normally

blocks transcription of the operon, but stops acting as a

repressor when lactose is present.

• The repressor protein senses lactose indirectly, through its

isomer allolactose.

• The lac operon contains three genes: lacZ, lacY, and lacA.

These genes are transcribed as a single mRNA, under control

of one promoter.

• Genes in the lac operon specify proteins that help the cell

utilize lactose.

o The lacZ gene encodes an enzyme called β-

galactosidase, which is responsible for splitting lactose

(a disaccharide) into readily usable glucose and

galactose (monosaccharides).

o The lacY gene encodes a membrane protein called

lactose permease, which is a transmembrane "pump" that

allows the cell to import lactose.

o The lacA gene encodes an enzyme known as a

transacetylase that attaches a particular chemical group

to target molecules.

• In addition to the three genes, the lac operon also contains a

number of regulatory DNA sequences. These are regions of

DNA to which particular regulatory proteins can bind,

controlling transcription of the operon.

• The promoter is the binding site for RNA polymerase, the

enzyme that performs transcription.

• The operator is a negative regulatory site bound by the lac

repressor protein. The operator overlaps with the

promoter, and when the lac repressor is bound, RNA

polymerase cannot bind to the promoter and start

transcription.

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Mechanism of

the operon in

the absence and

presence of

lactose.

In present of lactose

1. lactose converted to allolactose by transacetylase

allolactose bind to repressor protein and change the

confirmation of repressor protein.

Repressor protein cannot bind to operator

Lac operon swich on

2. RNA polymerase binds to the promoter and transcribes the

structural gene into mRNA.

3. mRNA is translated into three structural enzymes.

β-galactosidase hydrolyses lactose into glucose and

galactose

Permease transport lactose into E.coli

Transacetylase transfers an acetyl group from acetyl co-A to

β-galactosidase

In absent of lactose

1. Repressor protein binds to the operator and block promoter site

2. RNA polymerase cannot bind to the promoter to start

transcription.

3. Lac operon switches off.

chapter 6 : expression of biological information...ed the synthesis of short rna primer (using dna...

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