Gene expression concept and analysis

GeneExpression:ConceptandAnalysis
Noha Lotfy Ibrahim

The Central Dogma
• Proposed by Francis Crick 1958
• DNA holds the coded hereditary information in the
nucleus
• This code is expressed at the ribosome during
protein synthesis in the cytoplasm
• The protein produced by the genetic information is
what is influenced by natural selection
• If a protein is modified it cannot influence the gene
that codes for it
• Therefore there is one way flow of information:
DNA(transcription)  RNA(translation)  Protein

Gene Structure
 Gene is the sequence of nucleotides in DNA
encoding for one mRNA molecule or one
polypeptide chain.
 Eukaryotic gene structure: Most eukaryotic genes in
contrast to typical bacterial genes, the coding
sequences (exons) are interrupted by noncoding
DNA (introns). The gene must have (Exon; start
signals; stop signals; regulatory control elements).
 The average gene 7-10 exons spread over 10-16kb
of DNA.

Deoxy ribonuclic acid of DNA
• DNA is a very stable molecule
• It is a good medium for storing genetic
material but…
• DNA can do nothing for itself
• It requires enzymes for replication
• It requires enzymes for gene expression
• The information in DNA is required to
synthesise enzymes (proteins) but enzymes
are require to make DNA function

RIBONUCLEIC ACID (RNA)
•Found all over the cell
(nucleus, mitochondria, chloroplasts, ribosomes and
the soluble part of the cytoplasm).
•Certain forms of RNA have catalytic properties
•RIBOZYMES
•Ribosomes and snRNPs are ribozymes
•RNA could have been the first genetic information
synthesizing proteins…
•…and at the same time a biocatalyst
•Reverse transcriptase provides the possibility of
producing DNA copies from RNA

Types
• Messenger RNA (mRNA) <5%
• Ribosomal RNA (rRNA) Up to 80%
• Transfer RNA (tRNA) About 15%
• In eukaryotes small nuclear
ribonucleoproteins (snRNP).

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).

mRNA
• A long molecule 1 million Daltons
• Ephemeral
• Difficult to isolate
• mRNA provides the plan for the polypeptide
chain

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 for protein
synthesis

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

Gene Expression
 The process by which a gene's information is
converted into the structures and functions of a cell
by a process of producing a biologically functional
molecule of either protein or RNA (gene product) is
made (from genotype to phenotype).
• Gene expression is assumed to be controlled at
various points in the sequence leading to protein
synthesis.
• Idea: measuring amount of mRNA to see which genes
are expressed, as protein measuring is more difficult.

Gene Expression
Transcription
 Synthesis of mRNA that is complementary to one of
the strands of DNA. This happens in the nucleus of
eukaryotes.
Translation
 Ribosomes synthesize a polypeptide chain using the
genetic code on the mRNA molecule as its guide and
make protein according to its instruction.

Transcription plan
Transcription
DNA
messenger
RNA
Gene
Nucleus

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.

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)
• The parts which are edited out (by snRNP molecules)
are called INTRONS.
© 2010 Paul Billiet ODWS

Transcription Enzymes
RNA polymerase: The enzyme that controls
transcription and is characterized by:
 Search DNA for initiation site,
 It unwinds a short stretch of double helical DNA to
produce a single-stranded DNA template,
 It selects the correct ribonucleotide and catalyzes the
formation of a phosphodiester bond,
 It detects termination signals where transcript ends.
20

Transcription Enzymes
Polymerase I nucleolus Makes a large precursor to the
major rRNA (5.8S,18S and 28S
rRNA in vertebrates
Polymerase II nucleoplasm Synthesizes hnRNAs, which
are precursors to mRNAs. It
also make most small nuclear
RNAs (snRNAs
Polymerase III Nucleoplasm Makes the precursor to
5SrRNA, the tRNAs and
several other small cellular
and viral RNAs.

Transcription Factors
 Transcription factors are proteins that bind to
DNA near the start of transcription of a gene,
but they are not part of RNA polymerase
molecule .
 Transcription factors either inhibit or assist
RNA polymerase in initiation and maintenance
of transcription.
22

Regulatory elements
Eukaryotic Promoter
Conserved eukaryotic promoter elements Consensus sequence
CAAT box GGCCAATCT
TATA box TATAA
GC box GGGCGG
CAP site TAC
23
Eukaryotic Promoter lies adjacent to the gene, upstream
to the transcription startpoint, serve as a recognition
point that bind RNA polymerase (initiate transcription).
There are several different types of promoter found in
human genome, with different structure and different
regulatory properties class/I/II/III.

Enhancers
Enhancers are stretches of bases within DNA, about 50
to 150 base pairs in length; the activities of many
promoters are greatly increased by enhancers which
can exert their stimulatory actions over distances of
several thousands base pairs. It serves to increase the
efficiency of transcription, so increase the rate. It allow
RNA polymerase to bind DNA till reach the promotor.
24

Enhancers bind to transcription factors by at
Least 20 different proteins
Form a complex
change the configuration of the chromatin
folding, bending or looping of DNA.

Preinitiation Complex
 The general transcription factors combine with RNA
polymerase to form a preinitiation complex that is
competent to initiate transcription as soon as nucleotides
are available.
 The assembly of the preinitiation complex on each kind
of eukaryotic promoter (class II promoters recognized by
RNA polymerase II) begins with the binding of an
assembly factor to the promoter.
27

28
Source: http://www.news-medical.net/health/What-is-Gene-Expression.aspx

• The normal structure of the chromatin
suppresses the gene activity, making the
DNA relatively inaccessible to transcription
factors, and thus active transcription
complex can’t occur.
• Thus chromatin remodeling is needed
( it is a change in chromatin conformation in
which proteins of nucleosomes are released
from DNA , allowing DNA to be accessible for
TFs and RNA polymerase).

Inactive chromatin remodeled into active
chromatin by 2 biochemical modifications:
1. Acetylation of histone proteins by histone acetyl
transferases which loosen the association
between DNA and histone.
2. Specialised protein complexes disrupt the
nucleosome structure near the gene’s promoter
site.
This protein complex slides histone along DNA
transfer the histone to
other location on DNA molecule.

Active chromatin can be deactivated
by 3 biochemical reactions:
1. Histone deacetylation ( catalysed by histone
deacetylase).
2. Histone methylation ( catalysed by histone
methyl transferases).
3. Methylation of some DNA nucleotides by DNA
methyl transferases.
(Chromatin subjected to these modifications tends to
be transcriptionaly silent)

Initiation
 The polymerase binding causes the unwinding of the
DNA double helix which expose at least 12 bases on
the template.
 This is followed by initiation of RNA synthesis at this
starting point.
33

Initiation
 The RNA polymerase starts building the RNA chain;
it assembles ribonucleotides triphosphates: ATP;
GTP; CTP and UTP into a strand of RNA.
 After the first nucleotide is in place, the polymerase
joins a second nucleotide to the first, forming the
initial phosphodiester bond in the RNA chain.
34

Elongation
 RNA polymerase directs the sequential binding of
riboncleotides to the growing RNA chain in the 5' - 3'
direction.
 Each ribonucleotide is inserted into the growing RNA strand
following the rules of base pairing. This process is repeated
utill the desired RNA length is
synthesized……………………..
35

Termination
 Terminators at the end of genes; signal termination.
These work in conjunction with RNA polymerase to
loosen the association between RNA product and
DNA template. The result is that the RNA dissociate
from RNA polymerase and DNA and so stop
transcription.
 The product is immature RNA or pre mRNA (Primary
transcript).
36

Product of transcription
 The primary product of RNA transcription; the
hnRNAs contain both intronic and exonic sequences.
 These hnRNAs are processed in the nucleus to give
mature mRNAs that are transported to the cytoplasm
where to participate in protein synthesis.
37

RNA Processing (Pre-mRNA → mRNA)
 Capping
 Splicing
 Addition of poly A tail
38

RNA Processing
 Capping
 The cap structure is added to the 5' of the newly
transcribed mRNA precursor in the nucleus prior
to processing and subsequent transport of the
mRNA molecule to the cytoplasm.
 Splicing:
Step by step removal of pre mRNA and joining of
remaining exons; it takes place on a special
structure called spliceosomes.
39

RNA Processing
 Addition of poly A tail:
 Synthesis of the poly (A) tail involves cleavage of
its 3' end and then the addition of about 40- 200
adenine residues to form a poly (A) tail.
40

Alternative Splicing
 Alternative splicing: is a very common phenomenon
in higher eukaryotes. It is a way to get more than one
protein product out of the same gene and a way to
control gene expression in cells.
42

The Genetic Code
The sequence of codons in the mRNA defines
the primary structure of the final protein.
Three nucleotides in mRNA (a codon)specify
one amino acid in a protein.
44

The Genetic Code
 The triplet sequence of mRNA that specify certain
amino acid.
 There are only four letters to this code (A, G, C and U)
that represent 43= 64 different combination of bases;
61 of them code for 20 amino acids (AA); the last
three codon (UAG,UGA,UAA) don not code for
amino acids; they are termination codons.
 Degenerate
 More than one triplet codon specify the same amino
acid.
45

DNA & RNA Codon
DNA Codon RNA Codon
47

Translation plan
TRANSLATION
Complete protein
Polypeptide chain
Ribosomes
Stop codon Start codon

Translation
 Translation is the process by which ribosomes read
the genetic message in the mRNA and produce a
protein product according to the message's
instruction.
50

The protein synthesis occur in 3 phases
 Accurate and efficient initiation occurs; the
ribosomes binds to the mRNA, and the first amino
acid attached to its tRNA.
 Chain elongation, the ribosomes adds one amino acid
at a time to the growing polypeptide chain.
 Accurate and efficient termination, the ribosomes
releases the mRNA and the polypeptide.
51

Initiation
The initiation phase of protein synthesis requires over
10 eukaryotic Initiation Factors (eIFs): Factors are
needed to recognize the cap at the 5'end of an mRNA
and binding to the 40s ribosomal subunit.
Binding the initiator Met-tRNAiMet (methionyl-
tRNA) to the 40S small subunit of the ribosome.
52

Requirement for Translation
Ribosomes
tRNA
mRNA
 Amino acids
Initiation factors
Elongation factors
Termination factors
Aminoacyl tRNA synthetase enzymes:
Energy source
53

Initiation
Scanning to find the start codon by binding to the
5'cap of the mRNA and scanning downstream until they
find the first AUG (initiation codon).
The start codon must be located and positioned
correctly in the P site of the ribosome, and the initiator
tRNA must be positioned correctly in the same site.
Once the mRNA and initiator tRNA are correctly
bound, the 60S large subunit binds to form 80s initiation
complex with a release of the eIF factors.
54

Elongation
Transfer of proper aminoacyl-tRNA from
cytoplasm to A-site of ribosome;
Peptide bond formation; Peptidyl transferase forms
a peptide bond between the amino acid in the P site, and
the newly arrived aminoacyl tRNA in the A site. This
lengthens the peptide by one amino acids.
55

Elongation
Translocation; translocation of the new peptidyl t-
RNA with its mRNA codon in the A site into the free P
site occurs. Now the A site is free for another cycle of
aminoacyl t-RNA codon recognition and elongation.
Each translocation event moves mRNA, one codon
length through the ribosomes.
56

Termination
Translation termination requires specific protein
factors identified as releasing factors, RFs in E. coli and
eRFs in eukaryotes.
The signals for termination are the same in both
prokaryotes and eukaryotes. These signals are
termination codons present in the mRNA. There are 3
termination codons, UAG, UAA and UGA.
57

Termination
After multiple cycles of elongation and polymerization
of specific amino acids into protein molecules, a
nonsense codon = termination codon of mRNA appears
in the A site. The is recognized as a terminal signal by
eukaryotic releasing factors (eRF) which cause the
release of the newly synthesized protein from the
ribosomal complex.
58

Polysomes
 Most mRNA are translated by more than one
ribosome at a time; the result, a structure in which
many ribosomes translate a mRNA in tandem, is
called a polysomes.
59

Control of Gene Expression
 Transcriptional
 Posttranscriptional
 Translational
 Posttranslational
60

Control of gene expression
Control of gene expression depends various factors including:
Chromosomal activation or deactivation.
 Control of initiation of transcription.
 Processing of RNA (e.g. splicing).
 Control of RNA transport.
 Control of mRNA degradation.
 Control of initiation of translation (only in
eukaryotes).
 Post-translational modifications.
62

Summery: Eukaryotic Gene Expression
 Essentially all humans' genes contain introns. A notable
exception is the histone genes which are intronless.
 Eukaryote genes are not grouped in operons. Each
eukaryote gene is transcribed separately, with separate
transcriptional controls on each gene.
 Eukaryotic mRNA is modified through RNA splicing.
 Eukaryotic mRNA is generally monogenic
(monocistronic); code for only one polypeptide.
63

Summery: Eukaryotic Gene Expression
 Eukaryotic mRNA contain no Shine-Dalgarno
sequence to show the ribosomes where to start
translating. Instead, most eukaryotic mRNA have
caps at their 5` end which directs initiation factors to
bind and begin searching for an initiation codon.
 Eukaryotes have a separate RNA polymerase for each
type of RNA.
 In eukaryotes, polysomes are found in the cytoplasm.
 Eukaryotic protein synthesis initiation begins with
methionine not N formyl- methionine. 64

Prokaryotic vs. Eukaryotic
 Bacterial genetics are different.
 Prokaryote genes are grouped in operons.
 Prokaryotes have one type of RNA polymerase for all
types of RNA,
 mRNA is not modified
 The existence of introns in prokaryotes is extremely
rare.
65

Prokaryotic vs. Eukaryotic
To initiate transcription in bacteria, sigma factors bind
to RNA polymerases. RNA polymerases/ sigma factors
complex can then bind to promoter about 40
deoxyribonucleotide bases prior to the coding region of
the gene.
In prokaryotes, the newly synthesized mRNA is
polycistronic (polygenic) (code for more than one
polypeptide chain).
In prokaryotes, transcription of a gene and translation
of the resulting mRNA occur simultaneously. So many
polysomes are found associated with an active gene.
66

Gene Expression Analysis
• Polymerase Chain Reaction
• Quantitative PCR
• Microarray
• …….
• ……
67

Taq Polymerase
• Thermus aquaticus DNA
polymerase
• thermophilic organism
• enzymes resistant to high
temperatures
• 72-74o optimum
PCR Requirements
• heat-stable DNA
polymerase
• thermocycler
• target DNA and
primers

STEP TEMP TIME NOTES
Denature 94-96
o
0.5-2 min
longer:  denaturation,
but  enzyme, template
Annealing 15-25
o
< Tm 0.5-2 min
shorter:  specificity,
but  yield
Extension 72-75
o
~1 min (<kb)
Taq processivity = 150
nucleotides/sec
• mix DNA, primers,
dNTPs, Taq, buffer, Mg2+
• program thermocycler
for times and temps
–denaturation
–annealing
–extension
• 20-40 cycles
• analyze amplified DNA
(amplicons)
PCR Protocol

Disadvantage of traditional PCR
* Low sensitivity
* Short dynamic range
* Low resolution
* Non-automated
* Size-based discrimination only
* Results are not expressed as numbers
* Ethidium bromide staining is not very
quantitative
1. Why Real-time PCR ?

Advantages of real-time PCR
• amplification can be monitored real-time
• wider dynamic range of up to 1010-fold
• no post-PCR processing of products
(No gel-based analysis at the end of the PCR
reaction)
• ultra-rapid cycling (30 minutes to 2 hours)
• highly sequence-specific

1.It requires expensive equipments and
reagents
2.Due to its extremely high sensitivity,
you may get high deviations of the
same experiment, thus, the use of
internal control genes is a
recommended (in gene expression
experiments)
Disadvantages of real-time PCR

The QPCR Approach
Chemistry
l Use fluorescent dyes and probes
l Establish a linear correlation between PCR product and
fluorescence intensity
Detection
l Fluorescence detection to monitor amplification in real
time
Analysis
l Software for analysis and estimation of template
concentration
2- Theory of Real-time PCR ?

Concept of quantifications
• RT-PCR is identical to a standard PCR except
that the progress of RT-PCR is monitored by a
detector at each cycle.
• Each have used a kind of fluorescent marker
which binds to DNA.
• As the numbers of copies of genes increases;
the reaction of fluorescence increases.
• Quantifications is achieved by measuring the
increase of fluorescence during the
exponential phase of PCR.

qPCR detection-instrumention
How are the PCR product (amplicon) detected
in real time?
• By combining a PCR thermal cycler with a
fluorimeter
• qPCR instruments are commonly configured to
detect 2-5 different colors (or channels).
• Multiple detection channels allow for
quantifications of more than one target in one
single tube (multiplex qPCR).

cDNA microarray schema
From Duggan et al. Nature Genetics 21, 10 – 14 (1999)
color code for
relative expression

cDNA microarray raw data
Yeast genome microarray. The actual
size of the microarray is 18 mm by
18 mm. (DeRisi, Iyer & Brown,
Science, 268: 680-687, 1997)
• can be custom-made in
the laboratory
• always compares two
samples
• relatively cheap
• up to about 20,000
mRNAs measured per
array
• probes about 50 to a
few hundred nucleotides

Liver-enriched transcription factors
• Liver-specific gene expression is controlled
primarily at a transcriptional level.
• Transcriptional regulatory elements of genes
expressed in hepatocytes have identified
several liver-enriched transcription factors
(LETFs) which are key components of the
differentiation process for the fully functional
liver.

Glossary
 Alleles are forms of the same gene with small differences in their sequence of DNA bases.
 Alternative splicing: is a very common phenomenon in higher eukaryotes. It is a way to get more than one protein product out of
the same gene and a way to control gene expression in cells.
 Exon: a segment of a gene that is represented in the mature RNA product. Individual exons may contain coding DNAand/or
noncoding DNA (untranslated sequences).
 Bioinformatics I is the application of computer science and information technology to the field of biology and medicine
 Introns (intervening sequence) (A noncoding DNA sequence ): Intervening stretches of DNA that separate exons.
 Primary transcript: The initial production of gene transcription in the nucleus; an RNA containing copies of all exons and introns.
 RNA gene or non-coding RNA gene: RNA molecule that is not translated into a protein. Noncoding RNA genes produce
transcripts that exert their function without ever producing proteins. Non-coding RNA genes include transfer RNA (tRNA) and
ribosomal RNA (rRNA), small RNAs such as snoRNAs, microRNAs, siRNAsand piRNAs and lastly long ncRNAs.
 Enhancers and silencers: are DNA elements that stimulate or depress the transcription of associated genes; they rely on tissue
specific binding proteins for their activities; sometimes a DNA elements can act either as an enhancer or silencer depending on
what is bound to it.
 Activators: Additional gene-specific transcription factors that can bind to enhancer and help in transcription activation.
 Open reading frame (ORF): A reading frame that is uninterrupted by translation stop codon (reading frame that contains a start
codon and the subsequent translated region, but no stop codon).
 Directionality: in molecular biology, refers to the end-to-end chemical orientation of a single strand of nucleic acid. The chemical
convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5' end and a 3' end ( "five prime end"
and "three prime end"). The relative positions of structures along a strand of nucleic acid, including genes, transcription factors,
and polymerases are usually noted as being either upstream (towards the 5' end) or downstream (towards the 3' end).
 Reverse Transcription: Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA.
 Pseudogenes. DNA sequences that closely resemble known genes but are nonfunctional.
 More:http://www.ncbi.nlm.nih.gov/books/NBK7584/
81

Gene expression concept and analysis

Gene expression concept and analysis

Recommandé

Contenu connexe

Tendances

Tendances(20)

En vedette

En vedette(12)

Similaire à Gene expression concept and analysis

Similaire à Gene expression concept and analysis(20)

Dernier

Dernier(20)

Gene expression concept and analysis