How to Study DNA
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Transcript of 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
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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