Biologi Sel

DNA as the genetic material

H. J. Muller　postulated that genetic material, must

have three fundamental properties :
1.encode information for the production of compounds
that determine phenotypic features.
2.be able to replicate.
3.undergo change that can be perpetuated.
 DNA
• Functions
1. Storage of genetic information
2. Self-duplication & inheritance.
3. Expression of the genetic message.
• DNA’s major function is to code for proteins.
• Information is encoded in the order of the
nitrogenous bases.
 DNA
• DNA is a nucleic acid, made of repeating subunits called
nucleotides
• Nucleotides have three parts: a simple sugar, a phosphate
group, and a nitrogenous base.
Phosphate
group
Nitrogenous
base
Suga Nitrogenous
r base
Phosphat (A, G, C, or T)
e
group
Nucleotid
e

Thymine
(T)

Sugar
(deoxyribose
)

DNA
Polynucleotid Sugar-phosphate nucleotide
e backbone
DNA
• DNA has four kinds of bases, A, T, C, and G

Thymine (T) Cytosine (C) Adenine (A) Guanine (G)

Pyrimidines Purines
 The structure of nucleotides

Base pairing rule:

• A always pairs with T
• G always pairs with C
 RNA
• Different sugar (Ribose)
• U instead of T
• Single strand, usually
Nitrogenous base
(A, G, C, or U)

Phosphate
group

Uracil (U)

Sugar
(ribose)
DNA vs RNA
Nitrogenous base
(A, G, C, or U)

Phosphate
group

Uracil (U)

Sugar
(ribose)
RNA
 DNA is a double-stranded helix
• James Watson and Francis Crick worked out the
three-dimensional structure of DNA, based on work by
Rosalind Franklin
DNA and RNA
Chromosomes
 DNA and Chromosomes

Chromosome

E. Coli Bacterium

Bases on the
Chromosomes

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 Eukaryotic Chromosome Structure

Chromosome Nucleosome

DNA
double
helix

Coils
Supercoils

Histones

Copyright Pearson Prentice Hall

 Epigenetic
• The study of heritable changes in gene expression that
does not involve changes to the underlying DNA
sequence which in turn affects how cells read the
genes
• Influenced by several factors including age, the
environment/lifestyle, and disease state
• Epigenetic change can have more damaging effects
that can result in diseases like cancer, immunity and
neuropsychiatric disorder.
• DNA methylation, histone modification and non-coding
RNA (ncRNA)-associated gene silencing initiate and
sustain epigenetic change
 Chromosomes
Prokaryotic Eukaryotic
•Circular •Linear
•Very small •Fairly long
•1 chromosome per cell •Several chromosomes per
•Some enzymes and cell.
proteins are associated •Histone
with the DNA. proteins---”spools”. Same
•Not housed in a nucleus. in all eukaryotes
•Housed in a nucleus.
 Homologous Pairs
• two copies of every
chromosome
– one from mother
– one from father
• Homologous pairs
have the same genes
but not always the
same form of the
gene
The number of chromosomes per cell
varies in different species
 Genes
• Functional units
of DNA
• “Code” for
proteins
 Human Genome

• Refers to all the DNA

in human cells
• Contains 3 Billion
base pairs
• Sequencing (HGP)
completed April 25th
2003
 Genes and Proteins

• The sequence of nucleotides in DNA contain

information.
• This information is put to work through the
production of proteins.
• Proteins fold into complex, three dimensional shapes
to become key cell structures and regulators of cell
functions.
• DNA controls cells by encoding the instructions for
making proteins.
The central dogma of genetics
 DNA Replication
• Before a cell divides, it duplicates its DNA in a
process called replication.
• Replication ensures that each resulting cell will have
a complete set of DNA.
• Each strand of the DNA double helix has all the
information needed to reconstruct the other half by
the mechanism of base pairing.

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 DNA Replication Model
✓ Conservative
proposed both strands of one copy would be entirely
old DNA, while the other copy would have both
strands of new DNA.
✓ Dispersive
dsDNA might fragment, replicate dsDNA, and then
reassemble, creating a mosaic of old and new
dsDNA regions in each new chromosome.
✓ Semiconservative
DNA strands separate, and a complementary strand is
synthesized for each, so that sibling chromatids
have one old and one new strand.
 Fig. 3.1 Three models for the replication of DNA

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 DNA Replication
• In most prokaryotes, DNA replication begins at a
single point and continues in two directions.
• In eukaryotic chromosomes, DNA replication occurs
at hundreds of places. Replication proceeds in both
directions until each chromosome is completely
copied.
• The sites where separation and replication occur are
called replication forks.

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DNA Replication

Copyright Pearson Prentice Hall

Direction of DNA Replication
 Initiation of DNA Replication

1. Replication starts at origin of replication

2. Events in initiating DNA synthesis:
a. Gyrase (a type of topoisomerase) relaxes supercoils in
the region.
b. Initiator proteins attach.
c. DNA helicase binds initiator proteins on the DNA, and
denatures the region using ATP as an energy source.
d. DNA primase binds helicase to form a primosome, which
synthesizes a short RNA primer.
Initiation of Replication
Events occurring around a single replication fork
Events occurring around a single replication fork
 DNA Replication
• Helicase denaturing DNA causes tighter winding in other
parts of the circular chromosome. Gyrase relieves this
tension.
• Single-strand DNA-binding proteins (SSBs) bind the ssDNA
formed by helicase, preventing re-annealing.
• Primase synthesizes a primer on each template strand.
• DNA polymerase III adds nucleotides to the 3’ end of the
primer, synthesizing a new strand complementary to the
template.
• Leading strand is synthesized continuously, while lagging
strand is synthesized discontinuously, in the form of
Okazaki fragments. DNA replication is therefore
semi-discontinuous.
• Okazaki fragments are gradually joined together to make
a full-length dsDNA chromosome using DNA polymerase I
and DNA ligase
DNA Replication
DNA ligase sealing the gap
 DNA Replication in Eukaryotes

• DNA replication is very similar in both prokaryotes

and eukaryotes, except that eukaryotes have more
than one chromosome.
• Each eukaryotic chromosome must be copied, so
both DNA and histones must be doubled in each cell
cycle.
 Eukaryotic Replication Enzymes
Five DNA polymerases are known in mammalian cells:
– α (alpha) is nuclear, uses RNA primers, is involved in
nuclear DNA replication and has not been shown to
proofread.
– β (beta) is nuclear, serves in DNA repair and does not
show proofreading activity.
– δ (delta) is nuclear, uses RNA primers, is involved in
nuclear DNA replication and is capable of proofreading.
– γ (gamma) is mitochondrial, replicating the
mitochondrial DNA using RNA primers and proofreading.
– ε (epsilon) is nuclear, has proofreading activity and may
be used for DNA repair.
 Replicon

• The DNA replicated from a single origin is called a

replicon, or replication unit
• Replicon size is smaller in eukaryotes than
prokaryotes, and each chromosome contains
many replicons
 replication units
of eukaryotic chromosomes

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Replicating the Ends of Chromosomes

• When the ends of chromosomes are replicated and

the primers are removed from the 5’ ends, there is
no adjacent DNA strand to serve as a primer, and so
a single-stranded region is left at the 5’ end of the
new strand.
• If the gap is not addressed, chromosomes would
become shorter with each round of replication.
• This problem compensated by the addition of the
telomere repeats.
 The problem of replicating completely a linear chromosome in eukaryotes

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Synthesis of telomeric DNA by telomerase

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Transcription

RNA RNA nucleotide

polymerase

Direction of
transcription
Template
strand of DNA
Newly made RNA
Transcription
 RNA polymerase

• In transcription, DNA DNA of gene

helix unzips Promoter

DNA Terminator
DNA
Initiation
– RNA nucleotides line up
along one strand of
DNA, following the
base-pairing rules Elongation

– single-stranded
messenger RNA peels
away and DNA strands Termination
Growing
rejoin RNA

Completed RNA

RNA
polymerase
 The Stages of Transcription
■ Transcription occurs in three stages
■ Initiation

■ Elongation

■ Termination

51
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Transcription

52
Transcription
 Bases preceding the
Most of the promoter region is start site are
labeled with negative numbers, numbered in a
play a key role in transcription negative direction
There is no base
numbered 0

′ ′

′ ′

Sometimes termed the

Pribnow box, after its ′ ′
discoverer

Bases to the right are

numbered in a
positive direction

54
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Initiation σ factor

■ E. coli RNA polymerase

holoenzyme is composed of
Core enzyme and Sigma
factor
■ Sigma factor recognizes both
the –35 and –10 regions.
Binding of RNA polymerase to
the promoter forms the
closed complex.
■ The open complex is formed
when the TATAAT box in the σ factor is released, and the
core enzyme is able to
-10 region is unwound, a proceed
down the DNA.
short RNA strand is made, and
sigma factor is released at this
point σ factor

55
 Elongation
■ The RNA transcript is synthesized during the elongation
stage
■ The open complex formed by the action of RNA
polymerase is about 17 bases long, Behind the open
complex, the DNA rewinds back into a double helix
■ On average, the rate of RNA synthesis is about 43
nucleotides per second

56
 Termination
■ Occurs when the short RNA-DNA hybrid of the open
complex is forced to separate
■ This releases the newly made RNA as well as the RNA
polymerase
■ E. coli has two different mechanisms for termination
■ rho-dependent termination, requires a protein

known as r (rho)
■ rho-independent termination,does not require r

57
 Transcription in Eukaryotes

• The basic features of gene transcription are very similar

in bacteria and eukaryotes
• Gene transcription in eukaryotes is more complex
– Larger, more complex cells (organelles)
– Added cellular complexity means more genes that
encode proteins are required
– Multicellularity adds another level of regulation

58
 Eukaryotic RNA Polymerases
■ DNA is transcribed by three different RNA polymerases
■ RNA pol I, transcribes all rRNA genes (except for the 5S

rRNA)
■ RNA pol II

■ Transcribes all structural genes, synthesizes all

mRNAs
■ Transcribes some snRNA genes

■ RNA pol III

■ Transcribes all tRNA genes

■ And the 5S rRNA gene

59

© From Seth Darst, Bacterial RNA polymerase.
Current Opinion in Structural Biology.
Reprinted with permission of the author.
© From Patrick Cramer, David A. Bushnell, Roger D. Kornberg. "Structural Basis of
Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution." Science, Vol.
292:5523, 1863-1876, June 8, 2001.

′ ′
Structure of
RNA polymerase

′
′

61
 Eukaryotic Structural Genes
■ Eukaryotic promoter sequences are more variable and often
more complex than those of bacteria
■ For structural genes, at least three features are found in most
promoters
■ Regulatory elements

■ TATA box

■ Transcriptional start site

Usually an
adenine

62
 Transcription in Eukaryote
■ Proteins required for basal transcription to occur at the
promoter
■ RNA polymerase II

■ Five different proteins called general transcription factors

(GTFs)
■ A protein complex called mediator

63
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A closed complex

RNA pol II can now

proceed to the
Released after the elongation stage
open complex is
formed

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 Transcriptional termination
• Pre-mRNAs are modified by cleavage near their 3’ end
with subsequent attachment of a string of adenines
• Transcription terminates 500 to 2000 nucleotides
downstream from the poly A signal

66
Possible mechanisms for Pol II termination

′
′
′

′ ′

′ ′
′
′ ′ ′

′
′

67
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
 Eukaryotic RNA Processing
Exon Intron Exon Intron Exon
DNA
• Noncoding Transcription
Cap Addition of cap and tail
segments, RNA

introns, are transcript

with cap
and tail
Introns removed Tail

spliced out
• A cap and a tail mRNA
Exons spliced together

are added to Coding sequence

the ends NUCLEUS

CYTOPLASM
69
 Splicing
■ Three different splicing mechanisms have been identified
■ Group I intron splicing

■ Group II intron splicing

■ Spliceosome

■ All three cases involve

■ Removal of the intron RNA

■ Linkage of the exon RNA by a phosphodiester bond

70
 Benefits of Splicing
• Allows for genetic recombination
– Link exons from different genes together to create a new
mRNA
• Also allows for 1 primary transcript to encode for
multiple proteins by rearrangement of the exons
 Capping
■ Most mature mRNAs have a 7-methylguanosine covalently
attached at their 5’ end
■ The 7-methylguanosine cap structure is recognized by
cap-binding proteins
■ Cap-binding proteins play roles in the
■ Movement of some RNAs into the cytoplasm

■ Early stages of translation

■ Splicing of introns

72
 Tailing
■ Most mature mRNAs have a string of adenine nucleotides
at their 3’ ends. This is termed the polyA tail
■ The polyA tail is not encoded in the gene sequence It is
added enzymatically after the gene is completely
transcribed

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′ ′

Consensus
sequence in higher
eukaryotes
′

′
′ ′

Appears to be important in the

stability of mRNA and the
translation of the polypeptide
Length varies between species
From a few dozen adenines to
several hundred

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Eukaryotic vs
Procaryotic

• Procaryotic
– No CAP; have specific ribosome binding site upstream of AUG
– Polycistronic – multiple proteins from same mRNA
• Eucaryotic
– Monocistronic – one polypeptide per mRNA
Expression of • Some genes are
transcribed in large
Genes quantities because
we need large
amount of this
protein
• Some genes are
transcribed in small
quantities because
we need only a
small amount of
this protein
 Translation
• Translation is the process of translate mRNA to
protein
• Translate from one “language” (mRNA nucleotides)
to a second “language” (amino acids)
• Take place at ribosome in cytoplasm
Degenerate
DNA Code

• Nucleotides read 3 at a time meaning that there are

64 combinations for a codon (set of 3 nucleotides)
• Only 20 amino acids
– More than 1 codon per AA – degenerate code
with the exception of Met and Trp (least
abundant AAs in proteins)
Transfer RNA
• Translation requires an
Molecules adaptor molecule that
recognizes the codon on
mRNA and at a distant site
carries the appropriate
amino acid
• Intra-strand base pairing
allows for this
characteristic shape
• Anticodon is opposite
from where the amino
acid is attached
 Attachment of AA to tRNA

• Aminoacyl-tRNA synthase is the enzyme

responsible for linking the amino acid to the tRNA
• A specific enzyme for each amino acid and not for
the tRNA
 Ribosomal Subunits

• 1 large subunit – catalyzes the formation of the peptide bond

• 1 small subunit – matches the tRNA to the mRNA
• Moves along the mRNA adding amino acids to growing protein
chain
 Binding sites

• mRNA binding site

• Peptidyl-tRNA binding site (P-site), holds tRNA
attached to growing end of the peptide
• Aminoacyl-tRNA binding site (A-site), Holds the
incoming AA
• Exit site (E-site)
Ribosomal Assembly
Initiation Phase
• Initiation factors (IFs) catalyze the
steps – not well defined
• Step 1 – small ribosomal subunit
with the IF finds the start codon
–AUG
– Moves 5’ to 3’ on mRNA
– Initiator tRNA brings in the 1st AA
which is always Met and then can
bind the mRNA
• Step 2 – IF leaves and then large
subunit can bind – protein
synthesis continues
• Met is at the start of every protein
until post-translational
modification takes place
 Elongation Phase
• Step 1 – aminoacyl-tRNA comes into
empty A-site next to the occupied
P-site; pairs with the codon
• Step 2 – C’ end of peptide chain
uncouples from tRNA in P-site and
links to AA in A-site
– Peptidyl transferase responsible
for bond formation
– Each AA added carries the energy
for the addition of the next AA
• Step 3 – peptidyl-tRNA moves to the
P-site; requires hydrolysis of GTP
– tRNA released back to the
cytoplasmic pool
 Protein Release
• Protein released when a STOP
codon is encountered (UAG,
UAA, UGA)
• Cytoplasmic release factors bind
to the stop codon that gets to
the A-site; alters the peptidyl
transferase and adds H2O
instead of an AA
• Protein released and the
ribosome breaks into the 2
subunits to move on to another
mRNA
 Regulation of gene expression in
bacteria

• Natural selection has favored bacteria that produce

only the products needed by that cell
• A cell can regulate the production of enzymes by
feedback inhibition or by gene regulation
• Gene expression in bacteria is controlled by the
operon model
 Operons: The Basic Concept
• A cluster of functionally related genes can be under
coordinated control by a single “on-off switch”
• The regulatory “switch” is a segment of DNA called an
operator usually positioned within the promoter
• An operon is the entire stretch of DNA that includes the
operator, the promoter, and the genes that they control
• The operon can be switched off by a protein repressor
• The repressor prevents gene transcription by binding to
the operator and blocking RNA polymerase
• The repressor is the product of a separate regulatory
gene
General structure of an OPERON
Repressible and Inducible Operons: Two
Types of Negative Gene Regulation
• A repressible operon is one that is usually on; binding
of a repressor to the operator shuts off transcription
• The trp operon is a repressible operon
• An inducible operon is one that is usually off; a
molecule called an inducer inactivates the repressor
and turns on transcription
 trp operon
• By default the trp operon is on and the genes for
tryptophan synthesis are transcribed
• When tryptophan is present, it binds to the trp
repressor protein, which turns the operon off
• The repressor is active only in the presence of its
corepressor tryptophan; thus the trp operon is
turned off (repressed) if tryptophan levels are high
 trp
operon
Promoter Promoter
Genes of operon

DNA trpR trpE trpD trpC trpB trpA

Operator
Regulatory RNA
gene Start codon Stop codon
3′ polymerase
mRNA 5′
mRNA
5′
E D C B A

Protein Inactive Polypeptide subunits that make up

repressor enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on

DNA
No RNA
made

mRNA

Protein Active
repressor

Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off
 The lac operon
• The lac operon is an inducible operon and contains
genes that code for enzymes used in the hydrolysis
and metabolism of lactose
• By itself, the lac repressor is active and switches
the lac operon off
• A molecule called an inducer inactivates the
repressor to turn the lac operon on
 Regulatory Promoter
gene
Operator

DNA lacI lacZ

No
RNA
3′ made
mRNA
RNA
5′ polymerase

Active
Protein repressor

(a) Lactose absent, repressor active, operon off

lac operon

DNA lacI lacZ lacY lacA

RNA polymerase
3′
mRNA
mRNA 5′
5′

Protein β-Galactosidase Permease Transacetylase

Allolactose Inactive
(inducer) repressor

(b) Lactose present, repressor inactive, operon

on
Eukaryotic gene regulation occurs at
several levels
Control at DNA level by DNA methylation

◆ Small percentages of newly synthesized DNAs (~3%

in mammals) are chemically modified by
methylation.
◆ Methylation occurs most often in symmetrical CG
sequences.
◆ Transcriptionally active genes possess significantly
lower levels of methylated DNA than inactive genes.
◆ Methylation results in a human disease called fragile
X syndrome; FMR-1 gene is silenced by methylation.
 Control at transcription initiation
By using different sequences (promoter, enhancer or silencer
sequences) and factors, the rate of transcription of a gene is
controlled

gene X

promoter
 Control at mRNA stability
• The stem loop at 3’ end is an’ iron response element’.
• The stem loop is stabilised by a 90 kDa protein in the absence of
iron and protects the mRNA from degradation.
• In the presence of iron, transferrin receptor protein synthesis is
reduced.

aconitase)
 Control at mRNA stability

• Some hormones which enhance the production of

proteins also increase the half life of the protein’s
mRNA.

Estrogen : ovalbumin t1/2 from 2- 5hr to >24hr

Prolactin : casein t1/2 from 5 hr to 92hr

Control at initiation of translation

5’ UTR 3’ UTR
AUG UAA
• Bruce Alberts, Molecular Biology of the Cell,
6th Edition, Garland Science, New York
• Jack J. Pasternak, 2005, An Introduction to
Human Molecular Genetics Mechanisms of
Inherited Diseases 2nd Edition, Wiley, New
Jersey.