how to study dna

How to Study DNA

1. Genetic material

2. Expression product

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 Production of proteins requires two steps:steps:Transcription involves an enzyme (RNA Transcription involves an enzyme (RNA

polymerase) making an RNA copy of part of one polymerase) making an RNA copy of part of one DNA strand. DNA strand. There are four main classes of RNA:There are four main classes of RNA:i. i. Messenger RNAs (mRNA), which specify the amino acid Messenger RNAs (mRNA), which specify the amino acid sequence of a protein by using codons of the genetic sequence of a protein by using codons of the genetic code.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 eukaryotesiv. Small nuclear RNAs (snRNA), found only in eukaryotes ..

Translation converts the information in mRNA into Translation converts the information in mRNA into the amino acid sequence of a protein using the amino acid sequence of a protein using ribosomes, large complexes of rRNAs and proteins.ribosomes, large complexes of 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 production of the enzymes needed to metabolize lactose. Similar systems control metabolize lactose. Similar systems control many genes in bacteria and their viruses.many genes in 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 read to make a mRNA in a mRNA in the nucleus of the nucleus of our cellsour cells

Translation Translation – – Reading the Reading the mRNA to mRNA to make a make a protein in the protein in the cytoplasmcytoplasm

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

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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

Pol II Eukaryotic Promoter Elements

GC box~200 bp

CCAAT box~100 bp

TATA box~30 bp

Gene

Transcriptionstart site (TSS)

Exon Intron Exon

Pol II Eukaryotic Promoter Elements

Cap Region/Signaln C A G T n G

TATA box (~ 25 bp upstream)T A T A A A n G C C C

CCAAT box (~100 bp upstream)T A G C C A A T G

GC box (~200 bp upstream)A T A G G C G nGA

General modulators of General modulators of transcriptiontranscription

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

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 geneEukaryotic gene

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 Gene expression is regulated—not all genes

are constantly active and having their protein are constantly active and having their protein producedproduced

The regulation or feedback on gene The regulation or feedback on gene expression is how the cell’s metabolism is expression is how the cell’s metabolism is controlled. 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 When regulation of gene expression goes wrong—cancer!wrong—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

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

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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

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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

38

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)

Monocistronic 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