Gene:its nature expression and regulationPresentation Transcript
Gene Expression Overview
Salwa Hassan Teama
M.D. N.C.I. Cairo university
Gene ExpressionGene Expression
Eukaryotic CellEukaryotic Cell
The Gene StructureThe Gene Structure
Protein SynthesisProtein Synthesis
Prokaryotes Vs EukaryotesProkaryotes Vs Eukaryotes
Gene ExpressionGene Expression
Gene expressionGene expression is theis the
process by which a genesprocess by which a genes
information is converted intoinformation is converted into
the structures and functions ofthe structures and functions of
a cell by a process ofa cell by a process of
producing a biologicallyproducing a biologically
functional molecule offunctional molecule of eithereither
protein or RNA (gene product)protein or RNA (gene product)
is made.is made.
GeneGene expressionexpression isis
assumed to be controlled atassumed to be controlled at
various points in thevarious points in the sequencesequence
leading to proteinleading to protein synthesissynthesis..
Eukaryotic CellEukaryotic Cell
Gene StructureGene Structure
Eukaryotic gene structure:Eukaryotic gene structure:
MostMost eukaryotic genes ineukaryotic genes in
contrast to typicalcontrast to typical
bacterial genes,bacterial genes, thethe
coding sequencecoding sequence (exons)(exons)
are interrupted byare interrupted by
noncoding DNAnoncoding DNA (introns).(introns).
The gene must haveThe gene must have
( Exon; start signals; stop( Exon; start signals; stop
signals; regulatory controlsignals; regulatory control
Protein SynthesisProtein Synthesis
Protein SynthesisProtein Synthesis is theis the
process in whichprocess in which cellscells
buildbuild proteins fromproteins from
information in a DNAinformation in a DNA
gene in agene in a two majortwo major
synthesis of an RNAsynthesis of an RNA
(mRNA)(mRNA) that isthat is
complementary to one ofcomplementary to one of
the strands of DNA.the strands of DNA.
TranslationTranslation :: ribosomesribosomes
read a messenger RNAread a messenger RNA
and make proteinand make protein
according to itsaccording to its
RNA polymeraseRNA polymerase copies both the exons and the introns.copies both the exons and the introns.
The stretch of DNA that is transcribed into an RNA moleculeThe stretch of DNA that is transcribed into an RNA molecule
is called ais called a transcription unittranscription unit..
A transcription unit that is translated into proteinA transcription unit that is translated into protein containscontains
codingcoding sequence that is translated into protein andsequence that is translated into protein and
sequencessequences that direct and regulate protein synthesis;that direct and regulate protein synthesis;
Transcription proceeds in the 5' → 3' direction.Transcription proceeds in the 5' → 3' direction.
Transcription is divided into 3 phases: Initiation,Transcription is divided into 3 phases: Initiation, ElongationElongation
and Termination.and Termination...
RNA polymerase;RNA polymerase;
eukaryotic nuclei containeukaryotic nuclei contain
three RNA polymerasesthree RNA polymerases ..
RNA polymerase IRNA polymerase I isis
found in thefound in the nucleolusnucleolus;;
the otherthe other twotwo
polymerasespolymerases are locatedare located
in thein the nucleoplasmnucleoplasm. The. The
three nuclear RNAthree nuclear RNA
polymerase have differentpolymerase have different
roles in transcription.roles in transcription.
Polymerase IPolymerase I makes a largemakes a large
precursor to the major rRNAprecursor to the major rRNA
(5.8S,18S and 28S rRNA in(5.8S,18S and 28S rRNA in
Polymerase IIPolymerase II synthesizessynthesizes
hnRNAs, which are precursorshnRNAs, which are precursors
to mRNAs. It also maketo mRNAs. It also make
most small nuclear RNAsmost small nuclear RNAs
Polymerase IIIPolymerase III makes themakes the
precursor to 5SrRNA, theprecursor to 5SrRNA, the
tRNAs and several other smalltRNAs and several other small
cellular and viral RNAs.cellular and viral RNAs.
The general transcriptionThe general transcription
factors combine withfactors combine with
RNA polymeraseRNA polymerase toto
form a preinitiationform a preinitiation
complex that iscomplex that is
competent to initiatecompetent to initiate
transcription as soon astranscription as soon as
nucleotide are available.nucleotide are available.
The enzymeThe enzyme RNA polymeraseRNA polymerase recognizes arecognizes a promoter,promoter,
which lies upstream of the gene.which lies upstream of the gene. The polymeraseThe polymerase
binding causes thebinding causes the unwindingunwinding of the DNA double helix.of the DNA double helix.
This is followed by initiation of RNA synthesis at theThis is followed by initiation of RNA synthesis at the
starting point.starting point.
TheThe RNA polymeraseRNA polymerase starts building the RNA chain, itstarts building the RNA chain, it
assembles ribonucleotides triphosphates: ATP; GTP;assembles ribonucleotides triphosphates: ATP; GTP;
CTP and UTP into a strand of RNA.CTP and UTP into a strand of RNA.
After the first nucleotide is in place, the polymerase joinsAfter the first nucleotide is in place, the polymerase joins
a second nucleotide to the first, forming the initiala second nucleotide to the first, forming the initial
phosphodiester bond in the RNA chain.phosphodiester bond in the RNA chain.
RNA polymeraseRNA polymerase directs the sequential binding ofdirects the sequential binding of
riboncleotides to the growing RNA chain in the 5`-3`riboncleotides to the growing RNA chain in the 5`-3`
Each ribonucleotide is inserted into the growing RNAEach ribonucleotide is inserted into the growing RNA
strand following the rules of base pairing. This process isstrand following the rules of base pairing. This process is
repeated till the desired RNA length isrepeated till the desired RNA length is
Other regions at the end of genes; calledOther regions at the end of genes; called terminators,terminators,
signal termination. These work in conjunction with RNAsignal termination. These work in conjunction with RNA
polymerase to loosen the association between RNApolymerase to loosen the association between RNA
product and DNA template. The result is that the RNAproduct and DNA template. The result is that the RNA
dissociate from RNA polymerase and DNA and so stopdissociate from RNA polymerase and DNA and so stop
The product isThe product is immature RNA or pre mRNA (Primaryimmature RNA or pre mRNA (Primary
Pre-mRNA → mRNAPre-mRNA → mRNA
Capping:Capping: Synthesis of the cap. The 5` cap is a 7- methylguanosine (m7G)Synthesis of the cap. The 5` cap is a 7- methylguanosine (m7G)
. The cap protects the mRNA from being degraded by enzymes;. The cap protects the mRNA from being degraded by enzymes;
enhancement of mRNA translatability.enhancement of mRNA translatability.
SplicingSplicing:: Step-by-step removal of introns present in the pre-mRNA andStep-by-step removal of introns present in the pre-mRNA and
joining of the remaining exons. The removal of introns and joining of exonsjoining of the remaining exons. The removal of introns and joining of exons
takes place on a special structures called spliceosomes.takes place on a special structures called spliceosomes.
Polyadenylation:Polyadenylation: Synthesis of the poly (A) tail involves cleavage of its 3'Synthesis of the poly (A) tail involves cleavage of its 3'
end and then the addition of about 200end and then the addition of about 200 adenineadenine residues to form aresidues to form a poly (A)poly (A)
tail; This completes the mRNA moleculetail; This completes the mRNA molecule (mature mRNA),(mature mRNA), which is nowwhich is now
ready for export to the cytosol for protein synthesis.ready for export to the cytosol for protein synthesis.
RNA SplicingRNA Splicing
Alternative SplicingAlternative Splicing
Alternative splicing:Alternative splicing: is a very common phenomenon in higheris a very common phenomenon in higher
eukaryotes. It is a way to get more than one protein producteukaryotes. It is a way to get more than one protein product
out of the same gene and a way to control gene expressionout of the same gene and a way to control gene expression
in cells.in cells.
Translation is the process by which ribosomes read theTranslation is the process by which ribosomes read the
genetic message in the mRNA and produce a proteingenetic message in the mRNA and produce a protein
product according to the message's instruction.product according to the message's instruction.
RibosomesRibosomes are the siteare the site
ofof protein biosynthesisprotein biosynthesis
using the mRNA as ausing the mRNA as a
template, the ribosometemplate, the ribosome
traverses each codon oftraverses each codon of
the mRNA, pairing it withthe mRNA, pairing it with
the appropriate aminothe appropriate amino
acid. This is done usingacid. This is done using
molecules ofmolecules of transfertransfer
RNARNA (tRNA) containing a(tRNA) containing a
on one end and theon one end and the
appropriate amino acidappropriate amino acid
on the other.on the other.
Act asAct as adaptorsadaptors that can bind anthat can bind an
amino acid at one end and interactamino acid at one end and interact
with the mRNA at the other.with the mRNA at the other.
Source ofSource of coding informationcoding information for thefor the
protein synthesis system.protein synthesis system.
Contains start and stop signals forContains start and stop signals for
Eukaryotic mRNA isEukaryotic mRNA is cappedcapped. This is. This is
used as the recognition feature forused as the recognition feature for
ribosome binding.ribosome binding.
The site at which protein synthesisThe site at which protein synthesis
begins on the mRNA is especiallybegins on the mRNA is especially
crucial, since it sets thecrucial, since it sets the readingreading
frameframe for the whole length of thefor the whole length of the
messagemessage.. An error of one nucleotideAn error of one nucleotide
either way at this stage would causeeither way at this stage would cause
every subsequent codon in theevery subsequent codon in the
message to be misread, so that amessage to be misread, so that a
nonfunctional protein would result,nonfunctional protein would result,
the rate of initiation thus determinesthe rate of initiation thus determines
the rate at which the protein isthe rate at which the protein is
Amino acidsAmino acids are the monomersare the monomers
which arewhich are polymerizedpolymerized to produceto produce
proteinsproteins.. The amino acids areThe amino acids are
loaded ontoloaded onto tRNAtRNA molecules formolecules for
use in the process ofuse in the process of translationtranslation..
Initiation factorsInitiation factors help thehelp the
ribosome, initiator tRNA, and otherribosome, initiator tRNA, and other
components assemble the at thecomponents assemble the at the
correct location on the mRNA andcorrect location on the mRNA and
ensure that protein synthesisensure that protein synthesis
starts in the correct readingstarts in the correct reading
Elongation factorsElongation factors are responsibleare responsible
for moving the ribosome along thefor moving the ribosome along the
mRNA and maintain the correctmRNA and maintain the correct
reading framereading frame.. Facilitate removalFacilitate removal
ofof ""usedused"" tRNAs and bringing intRNAs and bringing in
Termination factorsTermination factors recognizerecognize
the stop codons and releasethe stop codons and release
proteins and ribosomes.proteins and ribosomes.
Aminoacyl tRNA synthetaseAminoacyl tRNA synthetase
enzymes:enzymes: It catalyze theIt catalyze the
covalent attachment of ancovalent attachment of an
amino acids to the end of theamino acids to the end of the
corresponding tRNA.corresponding tRNA.
Energy source:Energy source: ATP or GTPATP or GTP
which are synthesized in thewhich are synthesized in the
Preparatory steps forPreparatory steps for
protein synthesisprotein synthesis::
First,First, aminoacyl tRNAaminoacyl tRNA
synthetase join aminosynthetase join amino
acid to their specificacid to their specific
Second,Second, ribosomes mustribosomes must
dissociatedissociate into subunits atinto subunits at
the end of each round ofthe end of each round of
The protein synthesis occur in 3The protein synthesis occur in 3
11--Accurate and efficientAccurate and efficient initiationinitiation
occurs, the ribosomes binds tooccurs, the ribosomes binds to
the mRNA, and the first aminothe mRNA, and the first amino
acid attached to its tRNAacid attached to its tRNA..
22--ChainChain elongationelongation,, thethe
ribosomes adds one aminoribosomes adds one amino
acid at a time to the growingacid at a time to the growing
polypepyide chainpolypepyide chain..
33--Accurate and efficientAccurate and efficient
terminationtermination,, the ribosomesthe ribosomes
releases the mRNA and thereleases the mRNA and the
The initiation phase ofThe initiation phase of
protein synthesisprotein synthesis
requires over 10requires over 10
eukaryotic Initiationeukaryotic Initiation
Factors (eIFs):Factors (eIFs): FactorsFactors
are needed to recognizeare needed to recognize
the cap at the 5` end ofthe cap at the 5` end of
an mRNA and binding toan mRNA and binding to
the 40s ribosomalthe 40s ribosomal
Binding the initiator MetBinding the initiator Met--tRNAiMettRNAiMet
(methionyl- tRNA) to the 40S small(methionyl- tRNA) to the 40S small
subunit of the ribosomesubunit of the ribosome..
Scanning to find the start codonScanning to find the start codon byby
binding to the 5` cap of the mRNAbinding to the 5` cap of the mRNA
and scanning downstream until theyand scanning downstream until they
find the firstfind the first AUG (initiation codon)AUG (initiation codon)..
The start codon must be locatedThe start codon must be located
and positioned correctly in the Pand positioned correctly in the P
site of the ribosome and the initiatorsite of the ribosome and the initiator
tRNA must be positioned correctlytRNA must be positioned correctly
in the same site.in the same site.
Once the mRNA and initiator tRNAOnce the mRNA and initiator tRNA
are correctly bound, the 60S largeare correctly bound, the 60S large
subunit binds to form 80 s initiationsubunit binds to form 80 s initiation
complex with release of the eIFcomplex with release of the eIF
The large ribosomalThe large ribosomal
subunit contains threesubunit contains three
tRNA binding sites,tRNA binding sites,
designated A, P, and E.designated A, P, and E.
TheThe A siteA site binds anbinds an
aminoacyl-tRNA (a tRNAaminoacyl-tRNA (a tRNA
bound to an amino acid);bound to an amino acid);
thethe P siteP site binds abinds a
peptidyl-tRNA (a tRNApeptidyl-tRNA (a tRNA
bound to the peptidebound to the peptide
being synthesized); andbeing synthesized); and
thethe E siteE site binds a freebinds a free
tRNA before it exits thetRNA before it exits the
Transfer of properTransfer of proper
aminoacyl-tRNA fromaminoacyl-tRNA from
cytoplasm to A-site ofcytoplasm to A-site of
Peptide bond formation;Peptide bond formation;
Peptidyl transferasePeptidyl transferase
forms a peptide bondsforms a peptide bonds
between the amino acidbetween the amino acid
in the P site and thein the P site and the
newly arrived aminoacylnewly arrived aminoacyl
tRNA in the A site. ThistRNA in the A site. This
lengthens the peptide bylengthens the peptide by
one amino acids.one amino acids.
translocation of the newtranslocation of the new
peptidyl t-RNA with itspeptidyl t-RNA with its
mRNA codon in the A sitemRNA codon in the A site
into the free P site occursinto the free P site occurs
Now the A site is free forNow the A site is free for
another cycle ofanother cycle of
aminoacyl t-RNA codonaminoacyl t-RNA codon
recognition andrecognition and
elongation. Eachelongation. Each
translocation eventstranslocation events
moves mRNA , onemoves mRNA , one
codon length through thecodon length through the
Translational termination requires specific protein factorsTranslational termination requires specific protein factors
identified asidentified as releasing factors, RFsreleasing factors, RFs in E. coli and eRFs inin E. coli and eRFs in
The signals for termination are the same in both prokaryotesThe signals for termination are the same in both prokaryotes
and eukaryotes. These signals are termination codonsand eukaryotes. These signals are termination codons
present in the mRNA. There are 3present in the mRNA. There are 3 termination codons, UAG,termination codons, UAG,
UAA and UGA.UAA and UGA.
After multiple cycles ofAfter multiple cycles of
elongation andelongation and
polymerization of specificpolymerization of specific
amino acids into proteinamino acids into protein
molecules, a nonsensemolecules, a nonsense
codon = terminationcodon = termination
codon of mRNA appear incodon of mRNA appear in
the A site. The isthe A site. The is
recognized as terminalrecognized as terminal
signal by eukaryoticsignal by eukaryotic
releasing factors (eRF)releasing factors (eRF)
which cause the releasewhich cause the release
of the newly synthesizedof the newly synthesized
protein from theprotein from the
ribosomal complex.ribosomal complex.
Reading the instruction meansReading the instruction means
translating the code in the RNAtranslating the code in the RNA
((building block of DNA and RNA)building block of DNA and RNA)
toto amino acidsamino acids (building block of(building block of
Prokaryotic vs. EukaryoticProkaryotic vs. Eukaryotic
Eukaryotic DNA is wound aroundEukaryotic DNA is wound around histoneshistones to formto form
nucleosomesnucleosomes and packaged asand packaged as chromatinchromatin. Chromatin. Chromatin
has a strong influence on the accessibility of the DNA tohas a strong influence on the accessibility of the DNA to
transcription factorstranscription factors and the transcriptional machineryand the transcriptional machinery
includingincluding RNA polymerase.RNA polymerase.
Eukaryote genes are not grouped in operons. eachEukaryote genes are not grouped in operons. each
eukaryote gene is transcribed separately, with separateeukaryote gene is transcribed separately, with separate
transcriptional controls on each gene.transcriptional controls on each gene.
Protein synthesis takes place in the cytoplasm whileProtein synthesis takes place in the cytoplasm while
transcription and RNA processing take place in thetranscription and RNA processing take place in the
Essentially all humans' genes contain introns. A notableEssentially all humans' genes contain introns. A notable
exception is the histone genes which are intronless.exception is the histone genes which are intronless.
Prokaryotic vs. EukaryoticProkaryotic vs. Eukaryotic
Eukaryotic mRNA is modified throughEukaryotic mRNA is modified through RNA splicingRNA splicing..
Eukaryotic mRNA is generally monogenicEukaryotic mRNA is generally monogenic
(monocistronic); code for only one polypeptide.(monocistronic); code for only one polypeptide.
Eukaryotes have a separate RNA polymerase for eachEukaryotes have a separate RNA polymerase for each
type of RNA.type of RNA.
Eukaryotic mRNA containEukaryotic mRNA contain no Shine-Dalgarnono Shine-Dalgarno sequencesequence
to show the ribosomes where to start translating.to show the ribosomes where to start translating.
Instead, most eukaryotic mRNA have caps at their 5`Instead, most eukaryotic mRNA have caps at their 5`
end which directs initiation factors to bind and beginend which directs initiation factors to bind and begin
searching for an initiation codon.searching for an initiation codon.
Eukaryotic protein synthesis initiation begins withEukaryotic protein synthesis initiation begins with
methionine not N formyl- methionine.methionine not N formyl- methionine.
In eukaryotes, polysomes are found in the cytoplasm.In eukaryotes, polysomes are found in the cytoplasm.
Prokaryotic vs. EukaryoticProkaryotic vs. Eukaryotic
Bacterial genetics are different.Bacterial genetics are different.
Prokaryote genes are grouped in operons.Prokaryote genes are grouped in operons.
Prokaryotes have one type of RNA polymerase for allProkaryotes have one type of RNA polymerase for all
types of RNA,types of RNA,
mRNA is not modifiedmRNA is not modified
The existence of introns in prokaryotes is extremely rare.The existence of introns in prokaryotes is extremely rare.
To initiate transcription in bacteria, sigma factors bind toTo initiate transcription in bacteria, sigma factors bind to
RNA polymerases. RNA polymerases/ sigma factorsRNA polymerases. RNA polymerases/ sigma factors
complex can then bind to promoter about 40complex can then bind to promoter about 40
deoxyribonucleotide bases prior to the coding region ofdeoxyribonucleotide bases prior to the coding region of
the gene.the gene.
In prokaryotes, the newly synthesized mRNA isIn prokaryotes, the newly synthesized mRNA is
polycistronic (polygenic) (code for more than onepolycistronic (polygenic) (code for more than one
polypeptide chain).polypeptide chain).
In prokaryotes, transcription of a gene and translation ofIn prokaryotes, transcription of a gene and translation of
the resulting mRNA occur simultaneously. So manythe resulting mRNA occur simultaneously. So many
polysomes are found associated with an active gene.polysomes are found associated with an active gene.
References & Further Reading
Robert F.Weaver. Molecular Biology. Fourth Edition. Page 600. McGraw-Hill International Edition.Robert F.Weaver. Molecular Biology. Fourth Edition. Page 600. McGraw-Hill International Edition.
ISBN 978-0-07-110216-2ISBN 978-0-07-110216-2
InnisInnis,David H.,David H. GelfandGelfand,John J. Sninsky PCR Applications: Protocols for Functional Genomics: ISBN:0123721865,John J. Sninsky PCR Applications: Protocols for Functional Genomics: ISBN:0123721865
DanielDaniel H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. Washington, DC: AACC Press, 1996, ISBN 0-H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. Washington, DC: AACC Press, 1996, ISBN 0-
William B. Coleman,Gregory J. Tsongalis:William B. Coleman,Gregory J. Tsongalis: MolecularMolecular DiagnosticsDiagnostics: For the: For the ClinicalClinical LaboratorianLaboratorian:: ISBNISBN 1588293564...1588293564...
Robert F. Mueller,Ian D. Young.Robert F. Mueller,Ian D. Young. EmeryEmery''ss ElementsElements ofof MedicalMedical GeneticsGenetics: ISBN.: ISBN. 044307125X044307125X
DanielDaniel P.P. StitesStites,Abba T. Terr. Basic Human Immunology: ISBN.,Abba T. Terr. Basic Human Immunology: ISBN. 08385054300838505430
Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of theBruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the
cell. ISBN. 9780815341055cell. ISBN. 9780815341055
www.ebi.ac.uk/2can good introduction to bioinformatics and molecular biologywww.ebi.ac.uk/2can good introduction to bioinformatics and molecular biology
http://www.gene.ucl.ac.uk/nomenclature/guidelines.html defines the nomenclature for human geneshttp://www.gene.ucl.ac.uk/nomenclature/guidelines.html defines the nomenclature for human genes
Cell & Molecular Biology online: http://www.cellbio.com/recommend.htmlCell & Molecular Biology online: http://www.cellbio.com/recommend.html