The structure of DNAThe structure of DNA

Deoxyribonucleic acidDeoxyribonucleic acid DNA is made of nucleotidesDNA is made of nucleotides Each nucleotide is composed of phosphate, Each nucleotide is composed of phosphate,

sugar (deoxyribose) and a nitrogen basesugar (deoxyribose) and a nitrogen base 4 nitrogen bases – Adenine, Thymine, Guanine, 4 nitrogen bases – Adenine, Thymine, Guanine,

Cytosine (A,T,G,C)Cytosine (A,T,G,C) A-T, C-GA-T, C-G Bases are linked by hydrogen bondsBases are linked by hydrogen bonds

Figure 16.5 Sugar–phosphatebackbone

Nitrogenous bases

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)

Nitrogenous base

Phosphate

DNA nucleotide

Sugar(deoxyribose)

3 end

5 end

Figure 16.7

3.4 nm

1 nm

0.34 nm

Hydrogen bond

(a) Key features ofDNA structure

Space-fillingmodel

(c)(b) Partial chemical structure

3 end

5 end

3 end

5 end

T

T

A

A

G

G

C

C

C

C

C

C

C

C

C

C

C

G

G

G

G

G

G

G

G

G

T

T

T

T

T

T

A

A

A

A

A

A

Base PairingBase Pairing

DNA strandsDNA strands

Run in opposite directionsRun in opposite directions Helicase – unwinds helixHelicase – unwinds helix Topoisomerase – cuts and rejoins the helixTopoisomerase – cuts and rejoins the helix Ligase – brings together Okazaki fragmentsLigase – brings together Okazaki fragments DNA polymerases add nucleotides only to the DNA polymerases add nucleotides only to the

free 3free 3end of a growing strand; therefore, a end of a growing strand; therefore, a new DNA strand can elongate only in the new DNA strand can elongate only in the 55toto33directiondirection

Along one template strand of DNA, the DNA Along one template strand of DNA, the DNA polymerase synthesizes a polymerase synthesizes a leading strand leading strand continuously, moving toward the replication forkcontinuously, moving toward the replication fork

To elongate the other new strand, called the To elongate the other new strand, called the lagging strandlagging strand, DNA polymerase must work in , DNA polymerase must work in the direction away from the replication forkthe direction away from the replication fork

The lagging strand is synthesized as a series of The lagging strand is synthesized as a series of segments called segments called Okazaki fragmentsOkazaki fragments, which are , which are joined together by joined together by DNA ligaseDNA ligase

Figure 16.13

Topoisomerase

Primase

RNAprimer

Helicase

Single-strand bindingproteins

5

3

5

53

3

Origin of replication

RNA primer

Sliding clamp

DNA pol IIIParental DNA

3

5

5

33

5

3

5

3

5

3

5

Figure 16.15b

Figure 16.17

Overview

Leadingstrand

Origin of replication Lagging

strand

LeadingstrandLagging

strand Overall directionsof replicationLeading strand

DNA pol III

DNA pol III Lagging strand

DNA pol I DNA ligase

PrimerPrimase

ParentalDNA

5

5

5

5

5

33

3

333 2 1

4

Protein SynthesisProtein Synthesis

1.1. TranscriptionTranscription

The transfer of genetic info. From DNA to The transfer of genetic info. From DNA to messenger RNA (mRNA)messenger RNA (mRNA)

2.2. TranslationTranslation

The transfer of mRNA to proteinThe transfer of mRNA to protein

GenesGenes are pieces of DNA that code for proteins are pieces of DNA that code for proteins

mRNA – Uracil instead of ThyminemRNA – Uracil instead of Thymine

TranscriptionTranscription

DNA codes for single strand of mRNADNA codes for single strand of mRNA This happens in the nucleusThis happens in the nucleus RNA polymerase binds to the promoter RNA polymerase binds to the promoter

region on DNA templateregion on DNA template Sigma factor recognizes binding site on Sigma factor recognizes binding site on

DNADNA mRNA detatches at the terminator region mRNA detatches at the terminator region

of the DNA templateof the DNA template

TranscriptionTranscription

TranslationTranslation

The transfer of mRNA into a proteinThe transfer of mRNA into a protein This happens at the ribosomeThis happens at the ribosome Every 3 base pairs of mRNA is called a Every 3 base pairs of mRNA is called a

codoncodon tRNA hold anti-codons and amino acidstRNA hold anti-codons and amino acids tRNA bring amino acids down to the tRNA bring amino acids down to the

ribosomes using the corresponding anti-ribosomes using the corresponding anti-codon.codon.

TranslationTranslation

TranslationTranslation

The Genetic CodeThe Genetic Code

Discovery of DNA – Rosalind Discovery of DNA – Rosalind FranklinFranklin

Watson and CrickWatson and Crick

MutationsMutations

Sickle cell mutationSickle cell mutation

Prokaryotes vs. EukaryotesProkaryotes vs. Eukaryotes

In prokaryotes, translation of mRNA can In prokaryotes, translation of mRNA can begin before transcription has finishedbegin before transcription has finished

In a eukaryotic cell, the nuclear envelope In a eukaryotic cell, the nuclear envelope separates transcription from translation separates transcription from translation

Eukaryotic RNA transcripts are modified Eukaryotic RNA transcripts are modified through RNA processing to yield the finished through RNA processing to yield the finished mRNAmRNA

A A primary transcript primary transcript is the initial RNA is the initial RNA transcript from any gene prior to processingtranscript from any gene prior to processing

Comparing Gene Expression in Bacteria, Archaea, and Eukarya

• Bacteria and eukarya differ in their RNA polymerases, termination of transcription, and ribosomes; archaea tend to resemble eukarya in these respects

• Bacteria can simultaneously transcribe and translate the same gene

• In eukarya, transcription and translation are separated by the nuclear envelope

• In archaea, transcription and translation are likely coupled

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Figure 17.4

DNAtemplatestrand

TRANSCRIPTION

mRNA

TRANSLATION

Protein

Amino acid

Codon

Trp Phe Gly

5

5

Ser

U U U U U3

3

53

G

G

G G C C

T

C

A

A

AAAAA

T T T T

T

G

G G G

C C C G G

DNAmolecule

Gene 1

Gene 2

Gene 3

C C

Important vocabulary in Important vocabulary in transcriptiontranscription

The stretch of DNA that is transcribed is called a The stretch of DNA that is transcribed is called a transcription unittranscription unit

Transcription factors (sigma) – initiate the Transcription factors (sigma) – initiate the binding of the RNA polymerasebinding of the RNA polymerase

The completed assembly of transcription factors The completed assembly of transcription factors and RNA polymerase II bound to a promoter is and RNA polymerase II bound to a promoter is called a called a transcription initiation complextranscription initiation complex

A promoter called a A promoter called a TATA box TATA box is crucial in is crucial in forming the initiation complex in eukaryotesforming the initiation complex in eukaryotes

Figure 17.8

Transcription initiationcomplex forms

3

DNAPromoter

Nontemplate strand

53

53

53

Transcriptionfactors

RNA polymerase II

Transcription factors

53

53

53

RNA transcript

Transcription initiation complex

5 3

TATA box

T

T T T T T

A A A AA

A A

T

Several transcriptionfactors bind to DNA

2

A eukaryotic promoter1

Start point Template strand

RNA processing

• Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm

• During RNA processing, both ends of the primary transcript are usually altered

• Also, usually some interior parts of the molecule are cut out, and the other parts spliced together

• Each end of a pre-mRNA molecule is modified in a particular way

– The 5 end receives a modified nucleotide 5 cap

– The 3 end gets a poly-A tail• These modifications share several functions

– They seem to facilitate the export of mRNA to the cytoplasm

– They protect mRNA from hydrolytic enzymes– They help ribosomes attach to the 5 end

Figure 17.10

Protein-codingsegment

Polyadenylationsignal

5 3

35 5Cap UTRStartcodon

G P P P

Stopcodon

UTR

AAUAAA

Poly-A tail

AAA AAA…

RNA Splicing

• In some cases, RNA splicing is carried out by spliceosomes

• Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites

Figure 17.12-3RNA transcript (pre-mRNA)

5Exon 1

Protein

snRNA

snRNPs

Intron Exon 2

Other proteins

Spliceosome

5

Spliceosomecomponents

Cut-outintronmRNA

5Exon 1 Exon 2

GeneDNA

Exon 1 Exon 2 Exon 3Intron Intron

Transcription

RNA processing

Translation

Domain 3

Domain 2

Domain 1

Polypeptide

Figure 17.13

Figure 17.15

Amino acidattachmentsite

3

5

Hydrogenbonds

Anticodon

(a) Two-dimensional structure (b) Three-dimensional structure(c) Symbol used

in this book

Anticodon Anticodon3 5

Hydrogenbonds

Amino acidattachmentsite5

3

A A G

Figure 17.22

Ribosome

mRNA

Signalpeptide

SRP

1

SRPreceptorprotein

Translocationcomplex

ERLUMEN

2

3

45

6

Signalpeptideremoved

CYTOSOL

Protein

ERmembrane

What Is a Gene? Revisiting the Question

• The idea of the gene has evolved through the history of genetics

• We have considered a gene as– A discrete unit of inheritance

– A region of specific nucleotide sequence in a chromosome

– A DNA sequence that codes for a specific polypeptide chain

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Figure 17.26TRANSCRIPTION

DNA

RNApolymerase

ExonRNAtranscript

RNAPROCESSING

NUCLEUS

Intron

RNA transcript(pre-mRNA)

Poly-A

Poly-A

Aminoacyl-tRNA synthetase

AMINO ACIDACTIVATION

Aminoacid

tRNA

5 C

ap

Poly-A

3

GrowingpolypeptidemRNA

Aminoacyl(charged)tRNA

Anticodon

Ribosomalsubunits

A

AE

TRANSLATION

5 Cap

CYTOPLASM

P

E

Codon

Ribosome

5

3

Concept 17.5: Mutations of one or a few nucleotides can affect protein structure and function

• Mutations are changes in the genetic material of a cell or virus

• Point mutations are chemical changes in just one base pair of a gene

• The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein

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Figure 17.23

Wild-type hemoglobin

Wild-type hemoglobin DNA3

3

35

5 3

35

5553

mRNA

A AGC T T

A AGmRNA

Normal hemoglobin

Glu

Sickle-cell hemoglobin

Val

AA

AUG

GT

T

Sickle-cell hemoglobin

Mutant hemoglobin DNAC

Types of Small-Scale Mutations

• Point mutations within a gene can be divided into two general categories

– Nucleotide-pair substitutions

– One or more nucleotide-pair insertions or deletions

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Substitutions

• A nucleotide-pair substitution replaces one nucleotide and its partner with another pair of nucleotides

• Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code

• Missense mutations still code for an amino acid, but not the correct amino acid

• Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

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Insertions and Deletions

• Insertions and deletions are additions or losses of nucleotide pairs in a gene

• These mutations have a disastrous effect on the resulting protein more often than substitutions do

• Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation

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Wild type

DNA template strand

mRNA5

5

3

Protein

Amino end

A instead of G

(a) Nucleotide-pair substitution

3

3

5

Met Lys Phe Gly StopCarboxyl end

T T T T T

TTTTTA A A A A

AAAACC

C

C

A

A A A A A

G G G G

GC C

G GGU U U U UG

(b) Nucleotide-pair insertion or deletion

Extra A

35

53

Extra U

5 3

T T T T

T T T T

A

A A A

A

AT G G G G

GAAA

AC

CCCC A

T35

5 3

5T T T T TAAAACCA AC C

TTTTTA A A A ATG G G G

U instead of C

Stop

UA A A A AG GGU U U U UG

MetLys Phe Gly

Silent (no effect on amino acid sequence)

T instead of C

T T T T TAAAACCA GT C

T A T T TAAAACCA GC C

A instead of G

CA A A A AG AGU U U U UG UA A A AG GGU U U G AC

AA U U A AU UGU G G C UA

GA U A U AA UGU G U U CG

Met Lys Phe Ser

Stop

Stop Met Lys

missing

missing

Frameshift causing immediate nonsense(1 nucleotide-pair insertion)

Frameshift causing extensive missense (1 nucleotide-pair deletion)

missing

T T T T TTCAACCA AC G

AGTTTA A A A ATG G G C

Leu Ala

Missense

A instead of T

TTTTTA A A A ACG G A G

A

CA U A A AG GGU U U U UG

TTTTTA T A A ACG G G G

Met

Nonsense

Stop

U instead of A

35

35

53

35

53

35 3Met Phe Gly

No frameshift, but one amino acid missing(3 nucleotide-pair deletion)

missing

35

53

5 3U

T CA AA CA TTAC G

TA G T T T G G A ATC

T T C

A A G

Met

3

T

A

Stop

35

53

5 3

Figure 17.24

Chromosomal Alteration

• Deletion• Duplications – hemoglobin, antifreeze• Inversions• Translocations• Transposons – jumping genes (corn)

Gene Expression

• Gene expression is the act of going from genotype to phenotype

• DNA to mRNA to Protein• Genes are regulated by turning on and off

transcription

The lac operon in E.coli is the method by which E. coli make enzymes that metabolize lactose

Regulatory gene – produces the repressor

Repressor binds to the operator when lactose is absent

No Transcription – RNA polymerase cannot bind to the promoter

When lactose is present, it binds to the repressor pulling it off the operator

RNA polymerase binds the promoter – transcription begins

Lactose-digesting enzymes are made

(a) Lactose absent, repressor active, operon off

(b) Lactose present, repressor inactive, operon on

Regulatorygene

Promoter

Operator

DNA lacZlacI

lacI

DNA

mRNA5

3

NoRNAmade

RNApolymerase

ActiverepressorProtein

lac operon

lacZ lacY lacADNA

mRNA

5

3

Protein

mRNA 5

Inactiverepressor

RNA polymerase

Allolactose(inducer)

-Galactosidase Permease Transacetylase

Trp operon. E.coli will make tryptophan from scratch, but if it is in the surroundings, the E.coli will absorb it.

Different from the lac operon

Promoter

DNA

Regulatory gene

mRNA

trpR

5

3

Protein Inactive repressor

RNApolymerase

Promoter

trp operon

Genes of operon

Operator

mRNA 5

Start codon Stop codon

trpE trpD trpC trpB trpA

E D C B A

Polypeptide subunits that make upenzymes for tryptophan synthesis

(a) Tryptophan absent, repressor inactive, operon on

(b) Tryptophan present, repressor active, operon off

DNA

mRNA

Protein

Tryptophan (corepressor)

Activerepressor

No RNAmade

Proximal control elements are located close to the promoter

Distal control elements, groupings of which are called enhancers, may be far away from a gene or even located in an intron

Some transcription factors function as repressors, inhibiting expression of a particular gene by a variety of methods

A particular combination of control elements can activate transcription only when the appropriate activator proteins are present

ActivatorsDNA

EnhancerDistal controlelement

PromoterGene

TATA box

Generaltranscriptionfactors

DNA-bendingprotein

Group of mediator proteins

RNApolymerase II

RNApolymerase II

RNA synthesisTranscriptioninitiation complex

Controlelements

Enhancer Promoter

Albumin gene

Crystallingene

LIVER CELLNUCLEUS

Availableactivators

Albumin geneexpressed

Crystallin genenot expressed

(a) Liver cell

LENS CELLNUCLEUS

Availableactivators

Albumin genenot expressed

Crystallin geneexpressed

(b) Lens cell

Types of Genes Associated with Cancer

• Cancer can be caused by mutations to genes that regulate cell growth and division

• Tumor viruses can cause cancer in animals including humans

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Cancer Genes

• Oncogenes are cancer-causing genes• Proto-oncogenes are the

corresponding normal cellular genes that are responsible for normal cell growth and division

• Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle

Evidence That DNA Can Transform Bacteria

• The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928

• Griffith worked with two strains of a bacterium, one pathogenic and one harmless

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• When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic

• He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA

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Living S cells(control)

Living R cells(control)

Heat-killedS cells(control)

Mixture ofheat-killedS cells andliving R cells

Mouse dies Mouse diesMouse healthy Mouse healthy

Living S cells

EXPERIMENT

RESULTS

Figure 16.2

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Animation: Hershey-Chase ExperimentRight-click slide / select “Play”

Figure 16.4-3

Bacterial cell

Phage

Batch 1:Radioactivesulfur(35S)

Radioactiveprotein

DNA

Batch 2:Radioactivephosphorus(32P)

RadioactiveDNA

Emptyproteinshell

PhageDNA

Centrifuge

Centrifuge

Radioactivity(phage protein)in liquid

Pellet (bacterialcells and contents)

PelletRadioactivity(phage DNA)in pellet

EXPERIMENT