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’


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 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.


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


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’


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’



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’


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’



6.6. ProofreadingProofreadinginitial base-pairing errors are usually initial base-pairing errors are usually

corrected by DNA corrected by DNA polymerase.polymerase.


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


Inducer molecules→ Allolactose: - natural inducer, degradable IPTG (Isopropylthiogalactoside)- synthetic inducer, not metabolized

lacI repressor


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)


PSite

ASite

Largesubunit

Small subunit

mRNAmRNA


InitiationInitiation

mRNA


2-tRNA

G

aa2

A U

A

1-tRNA

U A C

aa1

anticodon

hydrogenbonds codon

mRNAmRNA


1-tRNA 2-tRNA

U A C G

aa1 aa2

A UA

anticodon

hydrogenbonds codon

peptide bond

3-tRNA

G A A

aa3

mRNAmRNA


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


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


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)


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


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


UAC

AUG

Tyr

GUA

CAU

Val

mRNA strand

3’

5’

HisMet Pro

GGA

CCU


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

Documents

How to Study Gene 1.Genetic Material 2.Expression Product