Integrative microRNA-gene expression network analysis in genetic ...
How to Study Gene 1.Genetic Material 2.Expression Product.
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Transcript of How to Study Gene 1.Genetic Material 2.Expression Product.
How to Study Gene
1. Genetic Material
2. Expression Product
DNA as Genetic MaterialDNA as Genetic MaterialDNA encodes all the information in the cellThe composition of the DNA is the same in
all cells within an organism– Variation among different cells is achieved by
reading the DNA differently
DNA contains four bases that encode all the information to make an organism’s life
DNADNADNA Consists of four kinds of
bases (A,C,G,T) joined to a sugar phosphate backbone
Bases carry the genetic information while the phosphate backbone is structural
Two complementary strands of bases (C-G) and (A-T)
DNA (DNA (Deoxyribonucleic Acid) Deoxyribonucleic Acid)
Deoxyribonucleotide
Deoxy Ribo Nucleotide
a Polymer of Deoxyribonucleotide Units
NCH
N
NHCN
NH2
O
H
H
HHO
H
H
OCH2PO
O
PO
O
P
O- O-O-
O-
O
(dATP)
Deoxyadenosine 5´-triphosphateO
H
H
HHO
H
H
NCH
N
NHCN
NH2
HOCH2
DeoxyRibonucleotide
DeoxyRibonucleosideDeoxyadenosine
OO=P-O O
Phosphate Group
NNitrogenous base (A, G, C, or T)
CH2
O
C1C4
C3 C2
5
Sugar(Deoxyribose)
DeoxyRibonucleotide
DeoxyRibonucleotide
5-carbon sugar (Deoxy ribose)Nitrogenous basePhosphate group
Backbone Sugar Molecules
Deoxyribose (DNA) Ribose (RNA)
O
OH
H
OH
H
H
H
HOCH2
HO
OH
OH
H
H
H
HOCH2
HO H
1´
2´3´
4´
5´
1´
2´3´
4´
5´
Ribose= Five Carbon Sugar Molecule
Deoxy ribo nucleotide
NITROGEN BASESNITROGEN BASES
HN
CHN
C
CN
CN
C
NH2
HHN
CHN
C
CN
CN
C
O
H2N
H
NC
CC
HN
C
O
CH3
HO
H
NC
CC
N
CH
O
H
H
NH2
Adenine Guanine
Thymine Cytosine
TwoPurines
TwoPyrimidines
9 9
1 1
It is composed of four different nitrogen bases
Nitrogenous BasesNitrogenous BasesPURINESPURINES
1.1. Adenine (A)Adenine (A)
2.2. Guanine (G)Guanine (G)
PYRIMIDINESPYRIMIDINES
3.3. Thymine (T)Thymine (T)
4.4. Cytosine (C)Cytosine (C) T or C
A or G
BASE-PAIRINGSBASE-PAIRINGSBaseBase
# of # of PurinesPurines PyrimidinesPyrimidines PairsPairs
H-BondsH-Bonds
Adenine (A)Adenine (A) Thymine (T)Thymine (T) A = TA = T 22
Guanine (G)Guanine (G) Cytosine (C)Cytosine (C) C GC G 3 3
CG
3 H-bonds
BASE-PAIRINGSBASE-PAIRINGS
CG
H-bonds
T A
Base Pairing Occurs Base Pairing Occurs Through Hydrogen BondsThrough Hydrogen Bonds
A-TG-C
Chargaff’s RuleChargaff’s Rule AdenineAdenine must pair with must pair with ThymineThymine
GuanineGuanine must pair with must pair with CytosineCytosine
Their amounts in a given DNA molecule Their amounts in a given DNA molecule will be will be about the sameabout the same..
G CT A
The DNA Backbone is a The DNA Backbone is a Deoxyribose PolymerDeoxyribose Polymer
Deoxyribose sugars are linked by Phosphodiester Bonds
O
P O
O
O-
H2C
O-
O-
OH
OH
H
H
HH
H2C
H2C
HH
H
H
OHH
O
O
P O
O
O
OP
O HH
H
H
OHH
O
HO
5´
3´
5´
5´
3´
3´
2´
2´
2´
1´
1´
1´
5´-p 3´-OH
5´ 3´
5´
3´
5´
3´ 5´
3´O
P O
O
O-
H2C
O-
O-
OH
OH
H
H
HH
H2C
H2C
HH
H
H
OHH
O
O
P O
O
O
OP
O HH
H
H
OHH
O
HO
5´
3´
5´
5´
3´
3´
2´
2´
2´
1´
1´
1´
O
P O
O
O-
H2C
O-
O-
OH
OH
H
H
HH
H2C
H2C
HH
H
H
OHH
O
O
P O
O
O
OP
O HH
H
H
OHH
O
HO
5´
3´
5´
5´
3´
3´
2´
2´
2´
1´
1´
1´
Base
Base
Base
O
P O
O
O-
H2C
O-
O-
OH
OH
H
H
HH
H2C
H2C
HH
H
H
OHH
O
O
P O
O
O
OP
O HH
H
H
OHH
O
HO
5´
3´
5´
5´
3´
3´
2´
2´
2´
1´
1´
1´
5´
3´ 5´
3´
NC
CC
N
CH
O
H
NH2
NC
CC
HN
C
O
CH3
HO
NC
CC
HN
C
O
CH3
HO
HO
OH
H
H
HHO
P O
O
O
OP
O
OH
H
H
HH
H2C
H2C
HH
H
H
HO
O-
O-
H2C
O-
O
OP
O
T
C
T
A
G
A
=
G C
A T
Double-stranded DNA Double-stranded DNA Forms a Double HelixForms a Double Helix
DNA Double HelixDNA Double Helix
NitrogenousNitrogenousBase (A,T,G or C)Base (A,T,G or C)
““Rungs of ladder”Rungs of ladder”
““Legs of ladder”Legs of ladder”
Phosphate &Phosphate &Sugar BackboneSugar Backbone
DNA Double HelixDNA Double Helix
P
P
P
O
O
O
1
23
4
5
5
3
3
5
P
P
PO
O
O
1
2 3
4
5
5
3
5
3
G C
T A
RIBO NUCLEIC ACIDRIBO NUCLEIC ACID
A polymer composed of nucleotides that A polymer composed of nucleotides that contain the sugar ribose and one of the four contain the sugar ribose and one of the four bases cytosine, adenine, guanine and bases cytosine, adenine, guanine and uracileuracile
Polynucleotide containing ribose sugar and Polynucleotide containing ribose sugar and uracile instead of thymineuracile instead of thymine
Genetic material of some virusesGenetic material of some virusesPrimary agent for transferring information Primary agent for transferring information
from the genome to the protein synthetic from the genome to the protein synthetic machinerymachinery
URACIL(U)
base with a single-ring structure
phosphate group
sugar (ribose)
Types of RNATypes of RNA
Three types ofThree types of RNARNA::
a)a) messenger RNA (mRNA)messenger RNA (mRNA)
b)b) transfer RNA (tRNA)transfer RNA (tRNA)
c)c) ribosome RNA (rRNA)ribosome RNA (rRNA)
Remember: all produced in the Remember: all produced in the nucleusnucleus
A. Messenger RNA (mRNA)A. Messenger RNA (mRNA)
Carries the information for a specific Carries the information for a specific protein.protein.
Made up of 500 to 1000 nucleotides long.Made up of 500 to 1000 nucleotides long.
Made up of codons (sequence of three Made up of codons (sequence of three bases: AUG - methionine).bases: AUG - methionine).
Each codon, is specific for an amino acid.Each codon, is specific for an amino acid.
A. Messenger RNA (mRNA)A. Messenger RNA (mRNA)
methionine glycine serine isoleucine glycine alanine stopcodon
proteinprotein
A U G G G C U C C A U C G G C G C A U A AmRNAmRNA
startcodon
Primary structure of a proteinPrimary structure of a protein
aa1 aa2 aa3 aa4 aa5 aa6
peptide bonds
codon 2 codon 3 codon 4 codon 5 codon 6 codon 7codon 1
B. Transfer RNA (tRNA)B. Transfer RNA (tRNA)
Made up of 75 to 80 nucleotides long.Picks up the appropriate amino acid
floating in the cytoplasm (amino acid activating enzyme)
Transports amino acids to the mRNA.Have anticodons that are complementary
to mRNA codons.Recognizes the appropriate codons on the
mRNA and bonds to them with H-bonds.
codon in mRNAanticodon
amino acid OH
amino acidattachment site
anticodon
tRNA molecules
amino acid attachment site
The structure of transfer RNA (tRNA)The structure of transfer RNA (tRNA)
Transfer RNA (tRNA)Transfer RNA (tRNA)
amino acidamino acidattachment siteattachment site
U A C
anticodonanticodon
methionineamino acidamino acid
C. Ribosomal RNA (rRNA)C. Ribosomal RNA (rRNA)
Made up of rRNA is 100 to 3000 nucleotides long.
Important structural component of a ribosome.
Associates with proteins to form ribosomes.
RibosomesRibosomes
Large and small subunits.Large and small subunits.
Composed of Composed of rRNA (40%) rRNA (40%) and and proteins (60%).proteins (60%).
Both units come together and help bind the Both units come together and help bind the mRNAmRNA and and tRNA.tRNA.
Two sites forTwo sites for tRNAtRNA
a.a. P site P site (first and last (first and last tRNA will attachtRNA will attach))
b.b. A site A site
RibosomesRibosomesOriginOrigin CompletComplet
e e ribosomribosomee
RibosomRibosomal al subunitsubunit
rRNA rRNA componentcomponentss
ProteinsProteins
Cytosol Cytosol (eukaryotic (eukaryotic ribosome)ribosome)
80 S80 S 40 S40 S
60 S60 S18 S18 S
5 S5 S
5.8 S5.8 S
25 S25 S
C.30C.30
C.50C.50
ChloroplastChloroplasts s (prokaryoti(prokaryotic c ribosome)ribosome)
70 S70 S 30 S30 S
50 S50 S16 S16 S
4.5 S4.5 S
5 S5 S
23 S23 S
C. 24C. 24
C. 35C. 35
MitochondMitochondrion rion (prokaryoti(prokaryotic c ribosome)ribosome)
78 S78 S 30 S30 S
50 S50 S18 S18 S
5 S5 S
26 S26 S
C. 33C. 33
C. 35C. 35
RibosomesRibosomes
PSite
ASite
Largesubunit
Small subunit
mRNAmRNA
A U G C U A C U U C G
Study of Genetic Study of Genetic MaterialMaterial
Number of chromosomes
Banding
Number of nucleotides
Sequencing
Structural genes Cloning
Non-structural genes Molecular marker
Central Dogma of Biology
DNA, RNA, and the Flow of Information
TranslationTranscription
Replication
Central Dogma (Modifications)
Transcription TranslationDNA
(1) Reverse transcription
Replication
RNA
(2)Self Replication
Protein
(3)Self Replication
(2)Ribozymes
DNA ReplicationDNA Replication
1. Origin of Replication
2. Strand Separation
3. Priming
4. Synthesis of new strand DNA
Origins of replicationOrigins of replication1.1. Replication Forks:
Hundreds of Y-shaped regions of replicating DNA
molecules where new strands are growing.
ReplicationReplicationForkFork
Parental DNA MoleculeParental DNA Molecule
3’
5’
3’
5’
DNA ReplicationDNA Replication
Origins of replicationOrigins of replication2.2. Replication Bubbles:Replication Bubbles:
a. Hundreds of replicating bubbles a. Hundreds of replicating bubbles (Eukaryotes).(Eukaryotes).
b. Single replication fork (bacteria).b. Single replication fork (bacteria).
Bubbles Bubbles
DNA ReplicationDNA Replication
DNA ReplicationStrand Separation:Strand Separation:Unwinding and separation of the parental double helix DNA
1. Helicase 1. Helicase Enzyme which catalyze the breaking H-Bonds between 2 nitrogen bases from different strand.2. Single-Strand Binding Proteins 2. Single-Strand Binding Proteins PProteins which attach and help keep the separated strands apart.
DNA ReplicationDNA Replication
Strand SeparationStrand Separation::
3.3. Topoisomerase Topoisomerase
enzyme which relieves stress on the DNA enzyme which relieves stress on the DNA molecule molecule
by allowing free rotation around a single by allowing free rotation around a single strand.strand.
Enzyme
DNA
Enzyme
DNA ReplicationDNA ReplicationPriming:Priming:The attachment of complementary primer on the
single stranded DNA
1.1. RNA primers RNA primers BBefore new DNA strands can form, there must be small pre-existing primers (RNA) present to start the addition of new nucleotides (DNA Polymerase).
2.2. PrimasePrimaseEEnzyme that polymerizes (synthesizes) the RNA Primer
DNA ReplicationDNA Replication
Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:The additional of nucleotide on RNA primer
1.1. DNA PolymeraseDNA Polymerasewith a RNA primer in place, DNA Polymerase with a RNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA (enzyme) catalyze the synthesis of a new DNA strand in the 5’ to 3’ directionstrand in the 5’ to 3’ direction..
RNARNAPrimerPrimerDNA PolymeraseDNA Polymerase
NucleotideNucleotide
5’
5’ 3’
DNA ReplicationDNA Replication
Synthesis of the new DNA StrandsSynthesis of the new DNA Strands
2.2. Leading StrandLeading Strand
synthesized as a single polymer in the 5’ synthesized as a single polymer in the 5’ to 3’ direction.to 3’ direction.
RNARNAPrimerPrimerDNA PolymeraseDNA PolymeraseNucleotidesNucleotides
3’5’
5’
DNA ReplicationDNA Replication
Synthesis of the new DNA StrandsSynthesis of the new DNA Strands
3.3. Lagging StrandLagging StrandIt also synthesized in the 5’ to 3’ direction, It also synthesized in the 5’ to 3’ direction,
but but discontinuously against overall direction discontinuously against overall direction of replication.of replication.
RNA PrimerRNA Primer
Leading StrandLeading Strand
DNA Polymerase
5’
5’
3’
3’
Lagging Strand
5’
5’
3’
3’
DNA ReplicationDNA Replication
Synthesis of the new DNA StrandsSynthesis of the new DNA Strands4.4. Okazaki FragmentOkazaki Fragment
series of short segments on the lagging series of short segments on the lagging strand.strand.
Lagging Strand
RNAPrimer
DNAPolymerase
3’
3’
5’
5’
Okazaki Fragment
DNA ReplicationDNA ReplicationSynthesis of the new DNA StrandsSynthesis of the new DNA Strands
5.5. DNA ligaseDNA ligasea linking enzyme that catalyzes the formation a linking enzyme that catalyzes the formation
of a covalent of a covalent bond bond from the 3’ to 5’ end of from the 3’ to 5’ end of joining stands.joining stands.
Example: joining two Okazaki fragments together.Example: joining two Okazaki fragments together.
Lagging Strand
Okazaki Fragment 2 2
DNA ligase
Okazaki Fragment 1
5’
5’
3’
3’
DNA ReplicationDNA Replication
Synthesis of the new DNA StrandsSynthesis of the new DNA Strands
6.6. ProofreadingProofreadinginitial base-pairing errors are usually initial base-pairing errors are usually
corrected by DNA corrected by DNA polymerase.polymerase.
DNA ReplicationDNA Replication
Semiconservative Model
Watson and Crick the two strands of the parental molecule separate, and each functions as a template
for synthesis of a new complementary strand.
Parental DNADNA Template
New DNA
DNA RepairDNA Repair
Excision repairExcision repair1.1.Damaged segment is excised by a repair Damaged segment is excised by a repair
enzyme (there are over 50 repair enzyme (there are over 50 repair enzymes).enzymes).
2.2.DNA polymerase and DNA ligase replace DNA polymerase and DNA ligase replace and bond the new nucleotides together.and bond the new nucleotides together.
TranscriptionTranslation
Gene ExpressionGene Expression
What is gene What is gene expression?expression?
The activation of a gene that results in a The activation of a gene that results in a protein.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 levels.
Expression of Genetic Expression of Genetic InformationInformation
Production of proteins requires two steps:Production of proteins requires two steps:Transcription involves an enzyme (RNA Transcription involves an enzyme (RNA
polymerase) making an RNA copy of part of polymerase) making an RNA copy of part of one DNA strand. There are four main one DNA strand. There are four main classes of RNA:classes of RNA:
i. i. Messenger RNAs (mRNA), which specify the Messenger RNAs (mRNA), which specify the amino acid sequence of a protein by using amino acid sequence of a protein by using codons of the genetic code.codons of the genetic code.
ii. Transfer RNAs (tRNA).ii. Transfer RNAs (tRNA).iii. Ribosomal RNAs (rRNA).iii. Ribosomal RNAs (rRNA).iv. Small nuclear RNAs (snRNA), found only in iv. Small nuclear RNAs (snRNA), found only in
eukaryoteseukaryotes..Translation converts the information in Translation converts the information in
mRNA into the amino acid sequence of a mRNA into the amino acid sequence of a protein using ribosomes, large complexes of protein using ribosomes, large complexes of rRNAs and proteins.rRNAs and proteins.
Expression of Genetic Expression of Genetic InformationInformation
Only some of the genes in a cell are active Only some of the genes in a cell are active at any given time, and activity also varies by at any given time, and activity also varies by tissue type and developmental stage. tissue type and developmental stage.
Regulation of gene expression is not Regulation of gene expression is not completely understood, but it has been completely understood, but it has been shown to involve an array of controlling shown to involve an array of controlling signals.signals.a. Jacob and Monod (1961) proposed the operon a. Jacob and Monod (1961) proposed the operon
model to explain prokaryotic gene regulation, model to explain prokaryotic gene regulation, showing that a genetic switch is used to control showing that a genetic switch is used to control production of the enzymes needed to metabolize production of the enzymes needed to metabolize lactose. Similar systems control many genes in lactose. Similar systems control many genes in bacteria and their viruses.bacteria and their viruses.
b. Genetic switches used in eukaryotes are b. Genetic switches used in eukaryotes are different and more complex, with much different and more complex, with much remaining to be learned about their functionremaining to be learned about their function..
Steps of gene expressionSteps of gene expression
TranscriptioTranscription – n – DNA is DNA is read to make a read to make a mRNA in the mRNA in the nucleus of our nucleus of our cellscells
Translation – Translation – Reading the Reading the mRNA to make mRNA to make a protein in a protein in the cytoplasmthe cytoplasm
59
TranscriptionTranscription DNA template: 3’-to-5’ DNA template: 3’-to-5’ RNA synthesis: 5’-3’; no primer neededRNA synthesis: 5’-3’; no primer needed
Structural genes: DNA that code for a specific polypeptide
(protein) Promoter : DNA segment that recognizes RNA
polymerase Operator : Element that serves as a binding site for an
inhibitor protein (modulator) that controls
transcription
Three (3) regulatory elements of transcription
61
Promoter RegionPromoter Region on DNA on DNA upstream from transcription start site initial binding site of RNA polymerase and initiation factors
(IFs) Promoter recognition: a prerequisite for initiation
Prokaryotic promoter regions
-10 site: “TATA” box-35 site = TTGACA
(TATA box)
Promoter RegionPromoter Region on DNA on DNA
Eukaryotic geneEukaryotic gene
Modulators of transcriptionModulators of transcription Modulators:Modulators:
(1) specificity factors, (2) repressors, (3) (1) specificity factors, (2) repressors, (3) activatorsactivators
1.1. Specificity factors:Specificity factors:Alter the specificity of RNA polymeraseAlter the specificity of RNA polymerase
s70 s32
Heat shock geneHousekeeping gene Heat shock promoter
Standard promoter
Modulators of transcriptionModulators of transcription
2. 2. Repressors: mediate negative gene regulation may impede access of RNA polymerase to the promoter actively block transcription bind to specific “operator” sequences (repressor
binding sites) Repressor binding is modulated by specific effectors
Coding sequence
Repressor
Operator
Promoter
Effector(e.g. endproduct)
Negative regulationNegative regulation
Repressor
EffectorExample: lac operon
RESULT:Transcription occurs when the gene is derepressed
Negative regulation
Repressor
Effector (= co-repressor)Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism
Modulators of transcriptionModulators of transcription
3.3. Activators:Activators: mediate positive gene regulation bind to specific regulatory DNA sequences (e.g.
enhancers) enhance the RNA polymerase -promoter interaction
and actively stimulate transcription common in eukaryotes
Coding sequence
Activator
promoter
RNA pol.
Positive regulation
RNA polymeraseActivator
Positive regulationPositive regulation
RNA polymerase
Activator
Effector
Gene expression takes place differently in prokaryotes and
eukaryotes. ProkaryotesProkaryotes
– No membrane No membrane bound organelles bound organelles (nucleus)(nucleus)
– More primitive More primitive organismsorganisms
– Only one circular Only one circular chromosomechromosome
– Bacteria are the Bacteria are the only organisms that only organisms that are prokaryotes.are prokaryotes.
EukaryotesEukaryotes– Membrane bound Membrane bound
organelles ( specialize organelles ( specialize in function –nucleus, in function –nucleus, mitochondria, mitochondria, chloroplast)chloroplast)
– Chromosomes are in Chromosomes are in pairs and not circularpairs and not circular
– All organisms that are All organisms that are not bacteria: protist, not bacteria: protist, fungi, plants and fungi, plants and animalsanimals
Prokaryotic gene organization
Prokaryotic transcriptional
regulatory regions
(promoters and operators) lie close to the
transcription start siteFunctionally
related genes are frequently located near each other
These “operons” are transcribed into a single mRNA with
internal translation
initiation sites
Prokaryotic Gene Prokaryotic Gene ExpressionExpression
PromoterCistron1Cistron2CistronNTerminator
Transcription RNA Polymerase
mRNA 5’ 3’
TranslationRibosome, tRNAs,Protein Factors
1 2 N
Polypeptides
NC
NC N
C
1 2 3
Expression mainly by controlling transcription
OperonsOperonsGenes that work together are located together A promoter plus a set of adjacent genes whose
gene products function together. They are controlled as a unit
They usually contain 2 –6 genes (up to 20 genes)
These genes are transcribed as a polycistronic transcript.
It is relatively common in prokaryotes It is rare in eukaryotes
Operon SystemOperon System
The lactose (lac) The lactose (lac) operonoperon
• Contains several elementsContains several elements– laclacZ gene = Z gene = ββ-galactosidase-galactosidase– laclacY gene = galactosidase permeaseY gene = galactosidase permease– laclacA gene = thiogalactoside transacetylaseA gene = thiogalactoside transacetylase– laclacI gene = I gene = lac lac repressorrepressor
– PPii = promoter for the = promoter for the laclacI geneI gene– P = promoter for P = promoter for laclac-operon-operon– QQ11 = main operator = main operator– QQ22 and Q and Q33 = secondary operator sites (pseudo- = secondary operator sites (pseudo-
operatorsoperators))
Pi P Z Y A I Q3 Q1 Q2
Regulation of the lac operonRegulation of the lac operon
Pi P Z Y A I Q3 Q1 Q2
Inducer molecules→ Allolactose: - natural inducer, degradable IPTG (Isopropylthiogalactoside)- synthetic inducer, not metabolized
lacI repressor
Pi P Z Y A I Q3 Q1 Q2
LacZ LacY LacA
The lac operon: model for gene expression
Includes three protein synthesis coding region--sometimes called "genes" as well as region of chromosome that controls transcription of genes Genes for proteins involved in the catabolism or breakdown of lactose When lactose is absent, no transcription of gene since no need for these proteinsWhen lactose is present, transcription of genes takes place so proteins are available to catalyze breakdown of lactose
Eukaryotic gene ExpressionEukaryotic gene Expression
1.Transcripts begin and end beyond the coding region
2.The primary transcript is processed by:5’ capping3’ formation / polyA
splicing
3.Mature transcripts are transported to the cytoplasm for translation
Regulation of gene expression
Plasmid
Gene (red) with an intron (green)Promoter
2. TranscriptionPrimary transcript
1. DNA replication
3. Posttranscriptional processing
4. Translation
mRNA degradation
Mature mRNA
5. Posttranslational processing
Protein degradationinactiveprotein
activeprotein
single copy vs. multicopy plasmids
Regulation of gene expression Gene expression is regulated—not all genes are Gene expression is regulated—not all genes are
constantly active and having their protein producedconstantly active and having their protein produced The regulation or feedback on gene expression is The regulation or feedback on gene expression is
how the cell’s metabolism is controlled. how the cell’s metabolism is controlled. This regulation can happen in different ways:This regulation can happen in different ways:
1. Transcriptional control (in nucleus):1. Transcriptional control (in nucleus):e.g. chromatin density and transcription factorse.g. chromatin density and transcription factors
2. Posttranscriptional control (nucleus)2. Posttranscriptional control (nucleus)e.g. mRNA processinge.g. mRNA processing
3. Translational control (cytoplasm)3. Translational control (cytoplasm)e.g. Differential ability of mRNA to bind ribosomese.g. Differential ability of mRNA to bind ribosomes
4. Posttranslational control (cytoplasm)4. Posttranslational control (cytoplasm)e.g. changes to the protein to make it functionale.g. changes to the protein to make it functional
When regulation of gene expression goes wrong—When regulation of gene expression goes wrong—cancer!cancer!
Transcription
Eukaryotic gene expression
Gene regulation of the transcription
Chr. I
Chr. II
Chr. III
Condition 1
“turned on”
“turned off”
Condition 2
“turned off”
“turned on”
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25 26
constitutively expressed gene
induced gene
repressedgene
inducible/ repressible genes
Gene regulationGene regulation
constitutively expressed gene
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25 26
Condition 3 Condition 4 upregulated gene expression
down regulated gene expression
DefinitionsDefinitionsConstitutively expressed genes
Genes that are actively transcribed (and translated) under all experimental conditions, at essentially all developmental stages, or in virtually all cells.
Inducible genesGenes that are transcribed and translated at higher levels in response to an inducing factor
Repressible genesGenes whose transcription and translation decreases in response to a repressing signal
Housekeeping genes –genes for enzymes of central metabolic pathways (e.g. TCA cycle)–these genes are constitutively expressed–the level of gene expression may vary
87
Post-Transcriptional Modification in EukaryotesPost-Transcriptional Modification in Eukaryotes
primary transcriptprimary transcript formed firstformed first then processed (3 steps) to form mature mRNA then processed (3 steps) to form mature mRNA then transported to cytoplasmthen transported to cytoplasm
Step 1: 7- methyl-guanosine “5’-cap” added to 5’ endStep 2: introns spliced out; exons link up
Step 3: Poly-A tail added to 3’ end
mature mRNA5’-cap- exons -3’ PolyA tail
88
Intron Splicing in EukaryotesIntron Splicing in Eukaryotes• Exons Exons : : coding regionscoding regions• Introns :Introns : noncoding regions noncoding regions • IntronsIntrons are removed by are removed by ““splicing”splicing”
AG at 3’ endof intron
GU at 5’ end
of intron
89
Splicesomes Roles in Splicing out Intron
RNA splicing occurs in small nuclear ribonucleoprotein RNA splicing occurs in small nuclear ribonucleoprotein particles (snRNPS) in spliceosomesparticles (snRNPS) in spliceosomes
90
5’ exon then moves to the 3’ splice acceptor site 5’ exon then moves to the 3’ splice acceptor site where a second cut is made by the spliceosomewhere a second cut is made by the spliceosome
Exon termini are joined and sealedExon termini are joined and sealed
Splicesomes Roles in Splicing out Intron
1 2
1 2
1 2
TranslationTranslation
Three parts:1. initiation: start codon (AUG)
2. elongation:3. termination: stop codon (UAG)
TranslationTranslation
PSite
ASite
Largesubunit
Small subunit
mRNAmRNA
A U G C U A C U U C G
InitiationInitiation
mRNA
A U G C U A C U U C G
2-tRNA
G
aa2
A U
A
1-tRNA
U A C
aa1
anticodon
hydrogenbonds codon
mRNAmRNA
A U G C U A C U U C G
1-tRNA 2-tRNA
U A C G
aa1 aa2
A UA
anticodon
hydrogenbonds codon
peptide bond
3-tRNA
G A A
aa3
mRNAmRNA
A U G C U A C U U C G
1-tRNA
2-tRNA
U A C
G
aa1
aa2
A UA
peptide bond
3-tRNA
G A A
aa3
Ribosomes move over one codon
(leaves)
mRNA
A U G C U A C U U C G
2-tRNA
G
aa1
aa2
A UA
peptide bonds
3-tRNA
G A A
aa3
4-tRNA
G C U
aa4
A C U
mRNAmRNA
A U G C U A C U U C G
2-tRNA
G
aa1aa2
A U
A
peptide bonds
3-tRNA
G A A
aa3
4-tRNA
G C U
aa4
A C U
(leaves)
Ribosomes move over one codon
mRNA
G C U A C U U C G
aa1aa2
A
peptide bonds
3-tRNA
G A A
aa3
4-tRNA
G C U
aa4
A C U
U G A
5-tRNA
aa5
mRNAmRNA
G C U A C U U C G
aa1aa2
A
peptide bonds
3-tRNA
G A A
aa3
4-tRNA
G C U
aa4
A C U
U G A
5-tRNA
aa5
Ribosomes move over one codon
mRNAmRNA
A C A U G U
aa1
aa2
U
primarystructureof a protein
aa3
200-tRNA
aa4
U A G
aa5
C U
aa200
aa199
terminatorterminator or stopor stop codoncodon
TerminationTermination
P Site A Site
E Site
Amino Acids forming Peptide chain
Ribosome
tRNA
anti-codon
codon
TranslationTranslation
UAC
AUG
Tyr
GUA
CAU
Val
mRNA strand
3’
5’
HisMet Pro
GGA
CCU
TranslationTranslation
The differenceThe difference• Eukaryotic and prokaryotic translation can react Eukaryotic and prokaryotic translation can react
differently to certain antibioticsdifferently to certain antibioticsPuromycinPuromycin
an analog tRNA and a general inhibitor of protein an analog tRNA and a general inhibitor of protein synthesissynthesis
CycloheximideCycloheximideonly inhibits protein synthesis by eukaryotic only inhibits protein synthesis by eukaryotic ribosomesribosomes
Chloramphenicol, Tetracycline, StreptomycinChloramphenicol, Tetracycline, Streptomycininhibit protein synthesis by prokaryotic ribosomeinhibit protein synthesis by prokaryotic ribosome
End ProductEnd Product
The end products of protein synthesis is a The end products of protein synthesis is a primary structure of a proteinprimary structure of a protein..
A sequence of A sequence of amino acid amino acid bonded together bonded together by by peptide bondspeptide bonds..
aa1
aa2 aa3 aa4aa5
aa200
aa199
PolyribosomePolyribosome
• Groups of ribosomes reading same Groups of ribosomes reading same mRNA mRNA simultaneously producing many simultaneously producing many proteins proteins (polypeptides).(polypeptides).
incominglarge
subunit
incomingsmall subunit polypeptide
mRNA1 2 3 4 5 6 7
Prokaryotes vs eukaryotes: key points
Prokaryotes Eukaryotes
Polycistronic mRNAs(single mRNA, multiple ORFs)
Moncistronic RNAs(One mRNA, one protein)
Operons(functional grouping)
No splicing
Ribosome scanningOften spliced
Regulatory sequences lie near (~100 bp) the start site
Regulatory sequences can be far (>1 kb) from the start site
Translation is concurrent with transcription
RNA processing is concurrent with transcription; translation occurs in a separate compartment
TYPES OF PROTEINSTYPES OF PROTEINS
Enzymes (Helicase)Enzymes (Helicase) Carrier (Haemoglobine)Carrier (Haemoglobine)
Immunoglobulin (Antibodies)Immunoglobulin (Antibodies) Hormones (Steroids)Hormones (Steroids) Structural (Muscle)Structural (Muscle)
Ionic (K+,Na+)Ionic (K+,Na+)
Coupled transcription and translation in bacteria
VALINE
HISTIDINE
LEUCINE
PROLINE THREONINE
GLUTAMATE
VALINE
original base triplet in a DNA strand
As DNA is replicated, proofreadingenzymes detect the mistake and
make a substitution for it:
a base substitution within the triplet (red)
One DNA molecule carries the original, unmutated sequence
The other DNAmolecule carries a gene mutation
POSSIBLE OUTCOMES:
OR
A summary of transcription and translation in a eukaryotic cellA summary of transcription and translation in a eukaryotic cell