Gene Expression: Concept and Analysis

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

The central dogma biology

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 andthe 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 informationsynthesizing proteins…

•…and at the same time a biocatalyst

•Reverse transcriptase provides the possibility ofproducing 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 andUracil (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 Daltonsa 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 Concept

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

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Transcription: The synthesis of a strand ofmRNA (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.

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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 TAC23

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.

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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.

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

• The normal structure of the chromatinsuppresses the gene activity, making theDNA relatively inaccessible to transcriptionfactors, and thus active transcriptioncomplex can’t occur.

• Thus chromatin remodeling is needed

( it is a change in chromatin conformation inwhich proteins of nucleosomes are releasedfrom DNA , allowing DNA to be accessible forTFs and RNA polymerase).

Inactive chromatin remodeled into active chromatin by 2 biochemical modifications:

1. Acetylation of histone proteins by histone acetyltransferases which loosen the associationbetween DNA and histone.

2. Specialised protein complexes disrupt thenucleosome structure near the gene’s promotersite.

This protein complex slides histone along DNA

transfer the histone toother 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)

Phases of transcription

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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.

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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.

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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……………………..

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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).

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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.

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RNA Processing (Pre-mRNA→ mRNA)

Capping

Splicing

Addition of poly A tail

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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.

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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.

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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.

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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.

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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.

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The Genetic Code

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DNA & RNA Codon

DNA Codon RNA Codon47

Translation plan

TRANSLATION

Complete protein

Polypeptide chain

Ribosomes

Stop codon Start codon

Translation

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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.

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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.

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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.

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Requirement for Translation

Ribosomes tRNAmRNA

Amino acids

Initiation factors

Elongation factors

Termination factors

Aminoacyl tRNA synthetase enzymes:

Energy source

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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.

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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.

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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.

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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.

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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.

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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.

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Control of Gene Expression

Transcriptional

Posttranscriptional

Translational

Posttranslational

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Control of Gene Expression

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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 ineukaryotes).

Post-translational modifications.

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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.

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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.

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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

• …….

• ……

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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 94-96o

0.5-2 minlonger: denaturation,

but enzyme, template

Annealing 15-25o < Tm 0.5-2 min

shorter: specificity,

but yield

Extension 72-75o

~1 min (<kb)Taq processivity = 150nucleotides/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. Why Real-time PCR ?

1.It requires expensive equipments andreagents

2.Due to its extremely high sensitivity,you may get high deviations of thesame experiment, thus, the use ofinternal control genes is arecommended (in gene expressionexperiments)

Disadvantages of real-time PCR

1. Why 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 controlledprimarily at a transcriptional level.

• Transcriptional regulatory elements of genesexpressed in hepatocytes have identifiedseveral liver-enriched transcription factors(LETFs) which are key components of thedifferentiation process for the fully functionalliver.

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 ofthe 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/ornoncoding 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 producetranscripts that exert their function without ever producing proteins. Non-coding RNA genes include transfer RNA (tRNA) andribosomal 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 tissuespecific binding proteins for their activities; sometimes a DNA elements can act either as an enhancer or silencer depending onwhat 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 startcodon 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 chemicalconvention 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/

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