GENE EXPRESSION:

REPLICATION,
TRANSCRIPTION &
TRANSLATION
Maya Dian Rakhmawatie
FK Unimus_
Blok 2 TA 2012/2013

Molecular Biology
Whats different from biology?
Study biology at molecular level,
overlaps with genetics and
biochemistry
Molecular Immunology
Molecular microbiology
Molecular pharmacology
2

Cell Theory
1. All organism composed of one or more
cells (prokaryotes/eukaryotes)
2. The cell is structural unit of life (gene?
DNA?
3. Cells can arise by division from a
preexisting cell (omnis cellulae cellula)
heredity?
3

What is a prokaryotes?
Eukaryotes?
Prokaryotes
No membrane bound
organelles (nucleus)
More primitive
organisms
Only one circular
chromosome
Bacteria are the only
organisms that are
prokaryotes.

Eukaryotes
Membrane bound
organelles ( specialize
in function nucleus,
mitochondria,
chloroplast)
Chromosomes are in
pairs and not circular
All organisms that are
not bacteria: fungi,
plants and animals
4

DNA
Deoxyribonucleic Acid
Double helix
Carries genetic
information
Located in the nucleus
The monomer is a
nucleotide
A phosphate
A ribose sugar
A nitrogenous base
5

What are the bases in DNA

A adenine
T thymine
C cytosine
G guanine
Base pair rules

Where is DNA located in the

Cell?

Where are the Genes Located?

Genes are located
on the
chromosomes.
Every species has
a different number
of chromosomes.
There are two
types of
chromosomes:
autosomes and sex
chromosomes

 Genes located on chromosomes (found in

the nucleus of a cell).
When a cell is undergoing cell
reproduction, the chromosomes are
visible.
Chromosomes appear when the chromatin
condenses and become visible.
(90%) the genetic material in the form of
chromatin.
A genome is the complete genetic
information contained in an individual.
(gene + chromosome)
9

What is Gene Expression?

Gene expression activation of a gene
results in a protein.
Biological processes, such as transcription,
and in case of proteins, also translation, that
yield a gene product.
A gene is expressed when its biological
product is present and active.
Gene expression is regulated at multiple
10

11

Regulation of Gene
Expression
Promoter Gene (red) with an intron (green)
1. DNA replication

Plasmid

single copy vs. multicopy

plasmids

2. Transcription
Primary
transcript
3. Posttranscriptional
processing

mRNA degradation

Mature
mRNA

4. Translation
inactive
protein
5. Posttranslational
processing

active
protein

Protein degradation

12

DNA Replication

Figure 17-1 Molecular Biology of the Cell ( Garland Science 2008)

13

Figure 17-3 Molecular Biology of the Cell ( Garland Science 2008)

14

Figure 17-4 Molecular Biology of the Cell ( Garland Science 2008)

15

The mammalian cell cycle

16

DNA Replication
Going ahead before meiosis and
mitosis
The goal is to make a copy of the
genetic information in the nucleus of
the cell so that the results of mitosis
and meiosis are the cells that have
the same genetic information as the
parent cell
17

DNA Replication
Need 4 type of nucleotida (DATP, DGTP,
DCTP, DTTP)
Need an enzyme:
a. Helicase, open double helix DNA into a
single chain DNA
b. Single strand binding-protein (SSB),
prevent the disintegration of a single chain
of DNA that will serve as a new DNA
template
18

DNA Replication
Need an enzyme:
c. Topoisomerase, loosen the tension that
existed at the DNA double chain winding
d. DNA polymerase, binding and combine
the nucleotida
e. DNA ligase, cover the single chain DNA
newly formed

19

Phase of DNA Replication

DNA replication starts from
disengagement of weak hydrogen bonds
between nitrogen base pairs in each DNA
chain, so that the two DNA strands are
separated.
Then the DNA polymerase molecule is
attached to the nitrogen bases apart and
start bonding with the nitrogen bases with
DNA nucleotides in nucleoplasma
accordance with the nitrogenous bases on
a single chain DNA template, which is A
20
attached to T, G-C, T-A, and C-G.

Phase of DNA Replication

Then the 3-OH group of new DNA nucleotides
react with the nucleophilic residue from new
nucleotide phosphate to form diester phosphate
bond.
After that, the DNA polymerase enzyme will shift
to the next DNA template and so on.
DNA nucleotides will be the same as the old DNA
template.
The replication process is read directions 3-5.
Once the replication is complete, the cell enters
21
G2 phase to mitosis or meiosis implement.

DNA Replication

22

DNA Replication

23

Control of Gene Expression

Controlling gene expression is often
accomplished by controlling
transcription initiation.
Regulatory proteins bind to DNA to
either block or stimulate transcription,
depending on how they interact with
RNA polymerase.
24

Control of Gene Expression

Prokaryotic organisms regulate gene
expression in response to their
environment.
Eukaryotic cells regulate gene
expression to maintain homeostasis
in the organism.

25

Expression of Genetic Information

Production of proteins requires two steps:
TRANSCRIPTION involves an enzyme (RNA polymerase) making an
RNA copy of part of one DNA strand.
There are five main classes of RNA:
a. Messenger RNAs (mRNA), duplicate from one chain of DNA,
which specify the amino acid sequence of a protein by using
codons of the genetic code.
b. Transfer RNAs (tRNA), synthesis from DNA, which bring an
amino acid (anti codon)
c. Ribosomal RNAs (rRNA), synthesis in nucleolus, bring to
ribosom for biosynthesis protein
d. Small nuclear RNAs (snRNA), found only in eukaryotes, break
intron and unification of exons
e. Heterogenous RNA (hnRNA), precursor mRNA
-

TRANSLATION converts the information in mRNA into the amino

acid sequence of a protein using ribosomes, large complexes of
rRNAs and proteins.
26

 Transcription is the
reading of the DNA and
changing the code to
mRNA.
Translation is
changing
the mRNA into a trait
by
using tRNA to interpret
the mRNA
27

Proteins Regulate Gene

Expression

28

Gene Structure Relevant to

Metabolic Regulation

29

 DNA in eukaryotes has regions of coding

and noncoding DNA.
The regions of DNA that code for proteins
or traits are called EXONS, while the
regions that do not code for proteins are
called INTRONS.

30

Eukaryotic Transcription
General transcription factors bind to
the promoter region of the gene.
RNA polymerase II then binds to the
promoter to begin transcription at
the start site (+1).
Enhancers are DNA sequences to
which specific transcription factors
(activators) bind to increase the
rate of transcription.
31

Intron Splicing in Eukaryotes

Exons: coding regions

Introns: noncoding regions
Introns are removed by splicing

GU
at 5 end
of intron

AG
at 3 end
of intron

32

Splicesomes Roles in Spicing

out Intron
RNA splicing occurs in small nuclear
ribonucleoprotein particles (snRNPS) in
spliceosomes

Spliceosom
es: protein
+ small
RNAs (U18)
complemen
tary to the
splice
junctions

indwiani@gmail.com

33

34

Eukaryotic Transcription
Coactivators and mediators
are also required for the
function of transcription factors.
coactivators and mediators bind to
transcription factors and bind to
other parts of the transcription
apparatus

35

36

Enhancer Control

Eukaryote genes
on a DNA strand
also have
noncoding control
sequences that
facilitate
transcription.
These are called
enhancers.
Transcription
factors are
additional proteins
that bind to RNA
polymerase and
enhancers
to help
37

 In prokayotes, transcription and translation occur

in the cytoplasm.
In eukaryotes, transcription occurs inside the
nucleus in a two step sequence of events.
Pre-mRNA includes both introns and exons for
the gene.
mRNA is only the coding portion (exons).
Translation occurs in the cytoplasm at the
ribosomes.
Reminder: The are three (5) types of RNA
Messenger (mRNA)
Transfer (tRNA)
Ribosomal (rRNA)
Small Nuclear (snRNA)
38
Heterogeneous (hnRNA)

RIBONUCLEIC ACID (RNA)

Found all over the cell
-nucleus,
-mitochondria,
-chloroplasts,
-ribosomes,
-the soluble part of the cytoplasm.
2007 Paul Billiet ODWS

39

Types of RNA

Messenger RNA (mRNA) <5%

Ribosomal RNA (rRNA) Up to 80%
Transfer RNA (tRNA) About 15%
In eukaryotes small nuclear
ribonucleoproteins (snRNP)
Heterogenous (hnRNA)
2007 Paul Billiet ODWS

40

Structural characteristics of
RNA molecules
Single polynucleotide strand
which may be looped or coiled (not a
double helix).
Sugar Ribose (not deoxyribose).
Bases used: Adenine, Guanine,
Cytosine and Uracil (not Thymine).

2007 Paul Billiet ODWS

41

mRNA

A long molecule 1 million Daltons

Ephemeral
Difficult to isolate
mRNA provides the plan for the
polypeptide chain

2007 Paul Billiet ODWS

42

rRNA
Coiled
Two subunits:
a long molecule 1 million Daltons
a short molecule 42 000 Daltons
Fairly stable
Found in ribosomes
Made as subunits in the nucleolus
rRNA provides the platform from
protein synthesis
2007 Paul Billiet ODWS

43

tRNA
Short molecule about 25 000 Daltons
Soluble
At least 61 different forms each has a
specific anticodon as part of its
structure.
tRNA translates the message
on the mRNA into a polypeptide
chain
2007 Paul Billiet ODWS

44

Transcription: The synthesis of a

strand of mRNA (and other RNAs)
Uses an enzyme RNA polymerase
Proceeds in the same direction as
replication (5 to 3)
Forms a complementary strand of mRNA
It begins at a promotor site which signals
the beginning of gene is not much further
down the molecule (about 20 to 30
nucleotides)
After the end of the gene is reached there
is a terminator sequence that tells RNA
polymerase to stop transcribing
NB
Terminator sequence terminator
codon
2007 Paul Billiet ODWS
45

Editing the mRNA

In prokaryotes the transcribed mRNA
goes straight to the ribosomes in the
cytoplasm
In eukaryotes the freshly transcribed
mRNA in the nucleus is about 5000
nucleotides long
When the same mRNA is used for
translation at the ribosome it is only 1000
nucleotides long
The mRNA has been edited
The parts which are kept for gene
expression are called EXONS (exons =
expressed)
2007
Paul Billiet ODWS
46
The parts which are edited out (by snRNP

Posttranscriptional
Regulation
Control of gene expression usually
involves the control of transcription
initiation.
But gene expression can be
controlled after transcription, with
mechanisms such as:
RNA interference
alternative splicing
RNA editing
mRNA degradation

47

Posttranscriptional
Regulation
RNA interference involves the use
of small RNA molecules
The enzyme Dicer chops double
stranded RNA into small pieces of
RNA
micro-RNAs bind to complementary
RNA to prevent translation
small interfering RNAs degrade
particular mRNAs before translation
48

Posttranscriptional
Regulation
Introns are spliced out of pre-mRNAs
to produce the mature mRNA that is
translated.
Alternative splicing recognizes
different splice sites in different
tissue types.
The mature mRNAs in each tissue
possess different exons, resulting in
different polypeptide products from
the same gene.
49

Posttranscriptional
Regulation
RNA editing creates mature mRNA
that are not truly encoded by the
genome.
For example
apolipoprotein B exists in 2 isoforms
one isoform is produced by editing the
mRNA to create a stop codon
this RNA editing is tissue-specific
50

Posttranscriptional
Regulation
Mature mRNA molecules have
various half-lives depending on the
gene and the location (tissue) of
expression.
The amount of polypeptide produced
from a particular gene can be
influenced by the half-life of the
mRNA molecules.
51

52

Translation
RNA
Single stranded
Does not contain
thymine but has uracil
instead.

tRNA carries 3 base

pair code for specific
amino acid.
Amino acids compose
polypeptid chains.
One or more
polypeptide chains
compose a protein
proteins provide the
blueprints for our
characteristics and

53

Transcription plan
Nucleus
Gene

DNA
Transcripti
on messenger
RNA

2007 Paul Billiet ODWS

54

Translation plan
Complete protein
Polypeptide chain
TRANSLATIO
N

Ribosomes

Stop codon
2007 Paul Billiet ODWS

Start codon
55

SKEMA TRANSLASI

56

Translation
Location: The ribosomes in the
cytoplasm that provide the
environment for translation
The genetic code is brought by the
mRNA molecule

2007 Paul Billiet ODWS

57

What is the genetic

code?
The genetic code consists of the
sequence of bases found along the
mRNA molecule
There are only four letters to this
code (A, G, C and U)
The code needs to be complex
enough to represent 20 different
amino acids used to build proteins
2007 Paul Billiet ODWS

58

indwiani@gmail.com

59

indwiani@gmail.com

60

How many combinations?

If one base represented one amino acid this
would only be able to produce
4 different combinations. (A, C, G and U)
If pairs of bases represented each amino acid
this would only be able to produce
4 x 4 = 16 combinations. (AA, AC, AG, AU, CA, CC,
CG, CU etc)
If triplets of bases represented each amino
acid, this would be able to produce
4 x 4 x 4 = 64 combinations
This is enough combinations to code for the 20
amino acids but is the code actually made of
triplets?
2007 Paul Billiet ODWS

61

Nature is logical!
Over 10 years biochemists
synthesised bits of mRNA with
different combinations
Then they used them to synthesise
polypeptides
The results proved the logical answer
was correct
The genetic code is made of triplets
of bases called codons
2007 Paul Billiet ODWS

62

Ribosomes initiate translation at ribosomebinding sites in polycistronic procaryotic mRNAs,

which can encode more than one protein

07_33_mRNA.encode.jpg

63

TRANSLATION PROCESS
INITIATION AUG
The next complementary
tRNA will bind at the
attachment binding
site(A) of the ribosome.
The adjacent amino acids
are then joined by
apeptide bondvia
apeptidaseenzyme.
Thus the polypeptide
chain begins to grow and
as it does, it is passed to
the next tRNA currently
occupying the64A site.

TRANSLATION PROCESS
ELONGATION
The ribosome then moves
1 codon down the mRNA
in a 5' to 3' direction.
This is achieved by
atranslocase enzyme.
The "old" tRNA is released
to bind another amino
acid and go in search of a
new codon. The binding of
a new aminoacid is
mediated by an enzyme
calledamino-acyl-tRNA
synthase
65

TRANSLATION PROCESS
TERMINATION
The process continues
along the mRNA until a
stop codon is reached.
While there is no tRNA
for a stop codon, there is
an enzyme
calledrelease
factorwhich cleaves
the polypeptide chain
resulting in a
newprotein.
66

07_35_polyribosome.jpg

A polyribosome
from a eucaryotic
cell

67

68

Signals Sequences

Presence of a sorting signal sequences in each of

the proteins directs them to a particular
compartment

Signal sequences direct proteins to the correct

destination compartment
15-60 amino acids long
Usually on the N-terminal end of the protein
Signal sequence is removed once the protein
transported

Protein Folding in Endoplasmic

Reticulum

 Disulfide bond

formation
proteins becomes
more stable
resistant to pH
changes or
enzymes
Glycosylation is a
covalent attachment of
oligosaccharides;
function:
Protects from
degradation
Transport signal for
packaging into
appropriate vesicle