biology-dna & protein synthesis chapter 10

8/8/2019 biology-DNA & protein synthesis Chapter 10

1/102

DNA AND PROTEINDNA AND PROTEIN

SYNTHESISSYNTHESISCHAPTER 10CHAPTER 10


2/102


3/102

Nucleic acidNucleic acid

Deoxyribonucleic acidDeoxyribonucleic acid

(DNA)(DNA)Ribonucleic acid (RNA)Ribonucleic acid (RNA)


4/102

10.110.1 DNA as the CarrierDNA as the Carrier

ofof Genetic MaterialGenetic Material


5/102

10.1.110.1.1 The Griffith experimentThe Griffith experiment

(transformation process)(transformation process)

Infected mice with 2 strain ofInfected mice with 2 strain of

pneumonococcuspneumonococcus bacteriabacteria

S strain : smooth colonies, ability to causeS strain : smooth colonies, ability to cause

disease, often death to host,disease, often death to host,

R strain : rough colonies, inability toR strain : rough colonies, inability toproduce pathogenic effectsproduce pathogenic effects


6/102

MiceMice ResultResult ConclusionConclusion

Live non-Live non-pathogenicpathogenic(R strain)(R strain)

SurvivedSurvived

(No living R(No living Rstrain in thestrain in theblood)blood)

SurvivedSurvived

(R strain does not(R strain does notcause pneumonia)cause pneumonia)

Live pathogenicLive pathogenic(S strain)(S strain)

DiedDied

(Living S strain(Living S strainin the blood)in the blood)

DiedDied

(S strain cause(S strain causepneumonia)pneumonia)

Heat killedHeat killedpathogenic strainpathogenic strain(S strain)(S strain)

SurvivedSurvived(No living S(No living Sstrain in thestrain in theblood)blood)

SurvivedSurvived(heat killed S Strain(heat killed S Straindoes not causedoes not causepneumonia)pneumonia)

Heat killedHeat killed

pathogenic (Spathogenic (Sstrain) + live nonstrain) + live nonpathogenic (Rpathogenic (Rstrain)strain)

Died. BloodDied. Blood

contains living Scontains living Sstrainstrain

Died. Blood contains SDied. Blood contains S

strain (a substancestrain (a substancefrom heat-killed Sfrom heat-killed Sstrain transform thestrain transform theharmless R strain intoharmless R strain intodeadly S strain)deadly S strain)


7/102

Living S cells

(control)

Living R cells

(control)

Heat-killed

S cells (control)

Mixture of heat-killed

S cells and living

R cells

Mouse dies

Living S cells

are found in

blood sample

Mouse healthy Mouse healthy Mouse dies

RESULTS


8/102Mouse lives Mouse livesMouse dies Mouse dies

R cells

injectedS cells

injectedHeat-killed

S cells

injected

R cells and

heat-killed S cells

injected


9/102

Griffith concluded that hereditary materialGriffith concluded that hereditary material

passed from the dead bacteria to the livingpassed from the dead bacteria to the livingbacteriabacteria

It changed non pathogenic strain to theIt changed non pathogenic strain to the

pathogenic strainpathogenic strain

The process is calledThe process is called transformationtransformation

Conclusion :Conclusion : Hereditary informationHereditary information can passcan passfrom dead cells to living ones, transformingfrom dead cells to living ones, transformingthem.them.


10/102

10.1.2The Avery experiment: evidence10.1.2The Avery experiment: evidence

that DNA carried geneticthat DNA carried genetic

information for transformationinformation for transformation prepares mixture of dead strainprepares mixture of dead strain

streptococcus (S strain) and living strain (Rstreptococcus (S strain) and living strain (R

strain) that Griffith used.strain) that Griffith used. Lysed (split open) S cells and separated cellLysed (split open) S cells and separated cell

content into several fractions (lipids,content into several fractions (lipids,

proteins, polysaccharides and nucleic acids)proteins, polysaccharides and nucleic acids)

Each fraction mix with R living cells to seeEach fraction mix with R living cells to see

transformation activitytransformation activity


11/102

RESULTSRESULTSFraction from SFraction from S

strain tested forstrain tested fortransformationtransformation

TransformationTransformation

activityactivity

PolysaccharidesPolysaccharides XX

Nucleic acids (DNANucleic acids (DNAand RNA)and RNA)

lipidslipids XX

proteinsproteins XX

Conclusion : DNA is the transforming principle

in bacteria


12/102

10.210.2 DNA replicationDNA replication

Hypothesis 1- Conservative replicationConservative replication

parental double helix would remain intact and generateparental double helix would remain intact and generateDNA copies consisting of entirely new moleculesDNA copies consisting of entirely new molecules


13/102

Hypothesis 2 - Dispersive replicationHypothesis 2 - Dispersive replicationparental DNA would become dispersed throughout theparental DNA would become dispersed throughout the

new copy so that each strand of all the daughternew copy so that each strand of all the daughtermolecules would be a mixture of old and new DNAmolecules would be a mixture of old and new DNA


14/102

Hypothesis 3 -Semiconservative replicationHypothesis 3 -Semiconservative replication

hydrogen bonds connecting base pairs arehydrogen bonds connecting base pairs are

disrupteddisrupted

2 polynucleotides chains unwind2 polynucleotides chains unwind

each chain acts as a template for the synthesiseach chain acts as a template for the synthesisof a new complementary polynucleotide chainof a new complementary polynucleotide chain

new nucleotides bind with the complementarynew nucleotides bind with the complementary

bases in each exposed chainbases in each exposed chain

2 identical molecules of DNA were form from2 identical molecules of DNA were form fromthe single parent moleculethe single parent molecule


15/102


16/102

10.2.1Meselsohn and Stahl experiment :10.2.1Meselsohn and Stahl experiment :

DNA replication is semiconservativeDNA replication is semiconservative

Grew bacteria in a medium containingGrew bacteria in a medium containing 1515NN

(heavy isotope of N). Bacteria incorporated(heavy isotope of N). Bacteria incorporated 1515NN

into their DNA and made the DNA denser thaninto their DNA and made the DNA denser thannormalnormal

Bacteria inBacteria in 1515N were transferred to aN were transferred to a 1414N andN and

allowed them to undergo additional cellallowed them to undergo additional cell

divisions (first generation)divisions (first generation)


17/102

Newly synthesized DNA strands less denseNewly synthesized DNA strands less dense

because they incorporated bases containingbecause they incorporated bases containing

lighterlighter1414N isotope.N isotope.

After another cycle cell division in theAfter another cycle cell division in the

medium containingmedium containing 1414N, 2 types of DNAN, 2 types of DNA

appeared. (exactly as predicted by theappeared. (exactly as predicted by the

semiconservative replication)semiconservative replication)

(d) H th i t ti


18/102

Bacteria are grown

in 15 N (heavy)

medium. All DNA

is heavy.

DNA is mixed with CsCl

solution, placed in an

ultracentrifuge, and

centrifuged at very

high speed for about 48 hours.

The greater concentration of

CsCl at the bottom of the

tube is due to sedimentation

under centrifugal force.

Some cells are

transferred to14 N (light)

medium.

First generation Second generation

Some cells

continue to

grow in 14 N

medium.

Cesium

chloride

(CsCl)

High

densityLow

density

DNA

(d) Hypothesis testing

(contd next slide)


19/102

15 N (heavy)

DNA

DNA molecules move to positions

where their density equals that of

the CsCl solution.

Two cell generations

after transfer to 14 N

One cell generation

after transfer to 14 N

Before transfer

to 14 N

14 N (light)

DNA

14 N 15 N

hybrid DNA

15 N (heavy)

DNA

14 N (light)

DNA14 N 15 N

hybrid DNA14 N 15 N

hybrid DNA

(e) Results

(contd)


20/102


21/102


22/102

TimeTime ObservationObservation

Before transferBefore transfer All dense. All strandsAll dense. All strandscontain heavy DNAcontain heavy DNA 1515 NN

First generationFirst generation Density of DNADensity of DNA

decreased to a valuedecreased to a valueintermediate betweenintermediate between1414 N-DNA andN-DNA and 1515 N DNAN DNA

Second generationSecond generation 2 density classes of2 density classes of

DNA observed. 1DNA observed. 1intermediate and 1intermediate and 1equal to that ofequal to that of1414 NNDNADNA


23/102

Interpreting the results:Interpreting the results:

11stst generation : Hybrid daughter DNA (generation : Hybrid daughter DNA (1515NN1414N)N)

22ndnd generation : Hybrid daughter DNA (generation : Hybrid daughter DNA (1515NN1414N) &N) &

light daughter DNA (light daughter DNA (1414NN1414N)N)

33rdrd generation : Hybrid daughter DNA (generation : Hybrid daughter DNA (1515NN1414N) &N) &

light daughter DNA (light daughter DNA (1414NN1414N)N)

Conclusion :Conclusion :

Replication is semiconservative.Replication is semiconservative.


24/102

(i)(i) DNA Helicase Separates Parental DNADNA Helicase Separates Parental DNA

StrandsStrands

Replication begins at special sites called origins ofReplication begins at special sites called origins of

replicationreplication

DNA helicase separates parental DNA, breaking HDNA helicase separates parental DNA, breaking H

bonds.bonds.

DNA separates, unwinds, forming replication bubbleDNA separates, unwinds, forming replication bubble

(has two replication forks)(has two replication forks)

Replication fork is formed at junction of single andReplication fork is formed at junction of single anddouble stranded regiondouble stranded region

Single stranded binding proteins bind to singleSingle stranded binding proteins bind to single

stranded DNA-prevents reformation of double helixstranded DNA-prevents reformation of double helix


25/102

Replication

Bubble

3

5

Replication Bubble

3

5


26/102

In eukaryotes, DNA replication begins at many sitesalong the giant DNA molecule of each chromosome.

Two daughter DNA molecules

Parental (template) strand

Daughter (new) strand0.25 m

Replication fork

Origin of replication

Bubble

In this micrograph, three replicationbubbles are visible along the DNAof a cultured Chinese hamster cell(TEM).


27/102

340 nm

Two replication forks

(b)

35

3

5Replication fork

Replication

bubbles

Single replication

bubble formed from

two merged

bubbles

(c)


28/102

DNA polymerase

DNA polymerase Origin of replication

Single-stranded

binding proteins

Direction of

replicationRNA primer

DNA

helicase

Twist introduced into

the helix by unwinding

3

5

3

5

3

5

35

3

5

3

5

35

3

5

3

3


29/102

(ii)(ii) DNA polymerase III synthesizes newDNA polymerase III synthesizes new

DNA strandsDNA strands

Each parental strand now serves as a template forEach parental strand now serves as a template for

new complementary strandnew complementary strand

DNA polymerase III synthesizes new strands atDNA polymerase III synthesizes new strands at

replication forkreplication fork

Synthesis proceeds in a 5 to 3 directionSynthesis proceeds in a 5 to 3 direction

DNA polymerase III only adds nucleotides to the 3DNA polymerase III only adds nucleotides to the 3

end of an existing polynucleotide chainend of an existing polynucleotide chain with bases that are complementary with templatewith bases that are complementary with template

nucleotides (Adenine (A) with Thymine (T), andnucleotides (Adenine (A) with Thymine (T), and

Guanine (G) with Cytosine (C)).Guanine (G) with Cytosine (C)).

LE 16-13


30/102

New strand

5 end

PhosphateBase

Sugar

Template strand

3 end 5 end 3 end

5 end

3 end

5 end

3 end

Nucleoside

triphosphate

DNA polymeraseIII

Pyrophosphate


31/102

Primase (RNA polymerase) synthesizes an RNAPrimase (RNA polymerase) synthesizes an RNAprimer at point where replication beginsprimer at point where replication begins

RNA primer RNA with 5 to 14 nucleotidesRNA primer RNA with 5 to 14 nucleotides

DNA polymerase III adds nucleotides to 3end ofDNA polymerase III adds nucleotides to 3end ofRNA primerRNA primer

DNA polymerase III catalayzes formation ofDNA polymerase III catalayzes formation ofphosphodiester bonds between nucleotides inphosphodiester bonds between nucleotides in

daughter DNAdaughter DNA


32/102

3

Leading strand

RNA primer

DNA polymerase III

Replication fork

Lagging strand

(first Okazaki fragment) Direction of

replication

DNA helix

5

3

5

3

5

3

5


33/102


34/102

(iii)(iii) DNA replication is continuous in oneDNA replication is continuous in one

strand and discontinuous in the otherstrand and discontinuous in the other

Leading strandLeading strand

- nucleotides added continuously and strand grows- nucleotides added continuously and strand grows

towards fork (same direction as helicase)towards fork (same direction as helicase) Lagging strandLagging strand

- nucleotides added in small segments called Okazaki- nucleotides added in small segments called Okazaki

fragments and grows away from fork (oppositefragments and grows away from fork (opposite

direction of helicase)direction of helicase) Each segment is initiated by a separate RNA primerEach segment is initiated by a separate RNA primer


35/102

Third Okazaki fragment

First Okazaki fragment


36/102


37/102

DNA polymerase I replaces RNA primer with DNADNA polymerase I replaces RNA primer with DNA

nucleotidesnucleotides Okazaki fragments are 100 to 2000 nucleotides inOkazaki fragments are 100 to 2000 nucleotides in

length and are joined together by DNA ligaselength and are joined together by DNA ligase

In eukaryotes, many replication bubbles formIn eukaryotes, many replication bubbles form

simultinaeuslysimultinaeusly Bubbles grow during replication and finally meetBubbles grow during replication and finally meet

each othereach other

DNA strands synthesized at bubble are joinedDNA strands synthesized at bubble are joined

together by DNA ligasetogether by DNA ligase

Each daughter DNA molecule consists of oneEach daughter DNA molecule consists of one

parental strand and one new strand.parental strand and one new strand.


38/102


39/102

Figure 11-13

Page 23135

3

5

3

5

3

5

3

5

3

53

5

35

3

5

3

5

3

5

+

+

DNA replication

Removal of primer

RNA primerRNA primer

(a)

(b)


40/102

T T G G G T T G G G G T T G G G G G T T G T T T T G G G G T T T T T G G G G G G

A A C C C A A C C C C A A C C C C C A A C A A A A C C C C A A A A AC C C C C C

5

3

3

5


41/102

The parent molecule hastwo complementarystrands of DNA. Each baseis paired by hydrogenbonding with its specificpartner, A with T and Gwith C.


42/102


The first step in replicationis separation of the twoDNA strands.


43/102

The parent molecule hastwo complementarystrands of DNA. Each baseis paired by hydrogenbonding with its specificpartner, A with T and G

with C.


Each parental strand nowserves as a template thatdetermines the order ofnucleotides along a new,complementary strand.


44/102



Each parental strand nowserves as a template thatdetermines the order ofnucleotides along a new,complementary strand.

The nucleotides areconnected to form thesugar-phosphate back-bones of the new strands.Each daughter DNAmolecule consists of oneparental strand and onenew strand.


45/102

10.310.3 The Concept of Gene and One-The Concept of Gene and One-

Gene/One- Polypeptide HypothesisGene/One- Polypeptide Hypothesis

Beadle & Tatum:Beadle & Tatum:Genes Specify EnzymesGenes Specify Enzymes

DNA (genes) contains information for synthesis ofDNA (genes) contains information for synthesis of

specific enzymesspecific enzymes If gene undergoes mutationIf gene undergoes mutation enzyme notenzyme not

synthesizedsynthesized molecules for growth notmolecules for growth notsynthesizedsynthesized

Thus, organism (mutant) cant grow unlessThus, organism (mutant) cant grow unlessprovided with the necessary molecules (forprovided with the necessary molecules (for

growth)growth)


46/102

Beadle & Tatum used the bread mold,Beadle & Tatum used the bread mold,NeurosporaNeurosporacrassacrassa because:because:

Have enzymes to build vitamin B6 and each of 20Have enzymes to build vitamin B6 and each of 20amino acids. Can grow on minimal mediumamino acids. Can grow on minimal medium(contains only sugar, ammonia, salts, a few(contains only sugar, ammonia, salts, a few

vitamin, and water.vitamin, and water. Can be bred in large numbers.Can be bred in large numbers.

Very short life cycle (10 days)Very short life cycle (10 days)

Has a haploid vegetative stage 1 set ofHas a haploid vegetative stage 1 set ofchromosomes for most of its life cyclechromosomes for most of its life cycle recessive genes not maskedrecessive genes not masked effect of mutationeffect of mutationseen immediately.seen immediately.


47/102

Wild typeWild type NeurosporaNeurospora spores exposed to X-ray.spores exposed to X-ray. DNA in some spores damaged in region codingDNA in some spores damaged in region coding

for synthesis of compound (s) necessary forfor synthesis of compound (s) necessary forgrowthgrowth

Therefore , some mold (mutant) cantTherefore , some mold (mutant) cantsynthesize this compound(s)synthesize this compound(s)

spores transferred to complete medium allspores transferred to complete medium allgrow. (Complete mediumgrow. (Complete medium contains allcontains allnecessary compounds for normal growth).necessary compounds for normal growth).


48/102

Isolating Growth-Deficient MutantsIsolating Growth-Deficient Mutants Individual cells of mold grown on minimalIndividual cells of mold grown on minimal

medium.medium.

Wild type strain (growth) and mutant strainWild type strain (growth) and mutant strain

(no growth) identified(no growth) identified


49/102

Identifying the DeficienciesIdentifying the Deficiencies Mutant strains transferred to a series ofMutant strains transferred to a series of

minimal medium.minimal medium.

A single different compound added to eachA single different compound added to each

mediummedium

medium in which growth occurredmedium in which growth occurred

contained compound that strain cantcontained compound that strain cant

synthesize.synthesize.

Expose Neurospora

spores to UV light or x rays


50/102

spores to UV light or x-rays

Each irradiated spore isused to establish culture on

complete growth medium(minimal medium plus

amino acids, vitamins, etc.)

Transfer cells tominimal medium

plus vitamins

Transfer cells tominimal mediumplus amino acids

Transfer cells to

minimal medium

(control)

Minimalmedium

plusarginine

Minimalmedium

plustryptophan

Minimalmedium

pluslysine

Minimalmedium

plusleucine

Minimalmedium

plus otheramino acids


51/102

One Gene/One-PolypeptideOne Gene/One-Polypeptide

synthesis of molecules involves series ofsynthesis of molecules involves series of

steps =biochemical pathways.steps =biochemical pathways.

Each step catalyzed by one enzyme.Each step catalyzed by one enzyme.

Beadle & Tatum:Beadle & Tatum: Genes encode enzymes.Genes encode enzymes.

Each gene located on different site onEach gene located on different site on

chromosome.chromosome. Mutation in a specific geneMutation in a specific gene synthesis ofsynthesis of

specific enzyme disruptedspecific enzyme disrupted no product.no product.

Example: Synthesis of arginine:Example: Synthesis of arginine:


52/102

Example: Synthesis of arginine:Example: Synthesis of arginine:

Gene AGene A Gene BGene B

OrnithineOrnithine ((Enzyme AEnzyme A)) CitrullineCitrulline ((Enzyme BEnzyme B)) ArginineArginine

If mutant mold can grow on mediumIf mutant mold can grow on medium

containing citrulline or arginine, but notcontaining citrulline or arginine, but notwith ornithine, mutation must havewith ornithine, mutation must haveoccurred atoccurred at gene Agene A (resulting in a(resulting in a ruinedruinedenzyme Aenzyme A).).

Each gene contains instruction for theEach gene contains instruction for thesynthesis of a single enzyme = Onesynthesis of a single enzyme = OneGene/One-Polypeptide (because enzymesGene/One-Polypeptide (because enzymes

contain polypeptide units).contain polypeptide units).


53/102


54/102


55/102

10.4 PROTEIN SYNTHESIS10.4 PROTEIN SYNTHESIS

Cells use information from DNA toCells use information from DNA to

synthesize proteinssynthesize proteins

Protein synthesized onProtein synthesized on ribosomesribosomes (RNA-(RNA-protein aggregates)protein aggregates)

Ribosome :Ribosome : Consists of large and small subunitsConsists of large and small subunits

Composed of RNA molecules & over 50 differentComposed of RNA molecules & over 50 different

proteinsproteins

3 sites for protein synthesis P, A, & E sites3 sites for protein synthesis P, A, & E sites


56/102


57/102

P site (Peptidyl-tRNA

binding site)

E site

(Exit site)

mRNA

binding site

A site (Aminoacyl-

tRNA binding site)

Large

subunit

Small

subunit

Schematic model showing binding sites

E P A

10 4 110 4 1 Kinds of RNAKinds of RNA


58/102

10.4.110.4.1 Kinds of RNAKinds of RNA Ribosomal RNA (rRNA)Ribosomal RNA (rRNA)

In ribosomesIn ribosomes

Function: ProvidesFunction: Provides sitesite for assembly offor assembly ofpolypeptidespolypeptides

Transfer RNA (tRNA)Transfer RNA (tRNA)

45 different types45 different types Functions:Functions:

Transport amino acidsTransport amino acids to ribosomes forto ribosomes forpolypeptide synthesispolypeptide synthesis

Positions each amino acid correctly onPositions each amino acid correctly on

polypeptide chainpolypeptide chain

Messenger RNA (mRNA)Messenger RNA (mRNA) RNA transcribed from DNARNA transcribed from DNA Function: DirectsFunction: Directspreciseprecise assembly of aminoassembly of amino

acidsacids into polypeptideinto polypeptide

h lTh C t l D


59/102

The Central DogmaThe Central Dogma

DNADNA

TranscriptionTranscription

mRNAmRNATranslationTranslation ProteinProtein

2 steps of Central Dogma =2 steps of Central Dogma = genegeneexpressionexpression..

TRANSCRIPTION DNA


60/102

RNA PROCESSING

RNAtranscript

5

Exon

NUCLEUS

FORMATION OFINITIATION COMPLEX

CYTOPLASM

3

RNApolymerase

RNA transcript(pre-mRNA)

Intron

Aminoacyl-tRNAsynthetase

Aminoacid

tRNA

AMINO ACID ACTIVATION

3

mRNA

A

P

E Ribosomalsubunits

5

Growingpolypeptide

E A

Activatedamino acid

Anticodon

TRANSLATION

Codon

Ribosome

10 4 2 Overview of Gene10 4 2 Overview of Gene


61/102

10.4.2 Overview of Gene10.4.2 Overview of GeneExpressionExpression

TranscriptionTranscription Transfer of information from DNA to RNA:Transfer of information from DNA to RNA:

RNA polymeraseRNA polymerase binds to promoterbinds to promoter

Enzyme moves along strandEnzyme moves along strand

Adds ribonucleotide to growing mRNA strandAdds ribonucleotide to growing mRNA strand

mRNA nucleotides are complementary to DNAmRNA nucleotides are complementary to DNAnucleotidesnucleotides

ExampleExample :: DNA - 3-ATTCGA-5DNA - 3-ATTCGA-5

mRNA - 5-UAAGCU-3mRNA - 5-UAAGCU-3

mRNA grows in 5 3 directionmRNA grows in 5 3 direction

Enzyme disengages from DNA at stop signalEnzyme disengages from DNA at stop signal mRNA separates from DNAmRNA separates from DNA


62/102

TranslationTranslation


63/102

Information from mRNA directs polypeptideInformation from mRNA directs polypeptidesynthesis by ribosomessynthesis by ribosomes

rRNA recognizes and binds to start sequence onrRNA recognizes and binds to start sequence onmRNAmRNA

Ribosomes move along mRNA, three nucleotides at aRibosomes move along mRNA, three nucleotides at atime specify one amino acidtime specify one amino acid

tRNA adds amino acids to growing polypeptide chaintRNA adds amino acids to growing polypeptide chain

Enzyme disengages from mRNA at stop signalEnzyme disengages from mRNA at stop signal

Polypeptide is releasedPolypeptide is released

Nontemplate strand


64/102

Translation

Codon 1 Codon 2 Codon 3 Codon 4 Codon 5 Codon 6

Polypeptide

Transcription

DNA

Template strand

mRNA

(complementary

copy of template

DNA strand)


65/102

10.4.3 The Genetic Code10.4.3 The Genetic Code

Code = 64 codons language for protein synthesisCode = 64 codons language for protein synthesis

Codon sequence of 3 consecutive mRNA bases whichCodon sequence of 3 consecutive mRNA bases whichspecifies an amino acid or signal to start/terminate thespecifies an amino acid or signal to start/terminate the

polypeptidepolypeptide Code is read continuously without gaps separating theCode is read continuously without gaps separating the

codons.codons.

Code isCode is degeneratedegenerate most amino acids have more than most amino acids have more than

one codonsone codons

Example: Leucine CUU, CUC, CUA, CUG, UUA, &Example: Leucine CUU, CUC, CUA, CUG, UUA, &UUGUUG

Certain codons are:Certain codons are: Start signal for initiation of polypeptide chain Start signal for initiation of polypeptide chain AUGAUG (also(also


66/102

Start signal for initiation of polypeptide chain Start signal for initiation of polypeptide chain AUGAUG (also(alsocodes for amino acid methionine)codes for amino acid methionine)

Stop signal for termination of polypeptide chain Stop signal for termination of polypeptide chain UAA,UAA,UAG, & UGAUAG, & UGA = = nonsense codonsnonsense codons

Code is universal same inCode is universal same in almost allalmost all organismsorganisms Importance of universality:Importance of universality:

Evidence of common evolutionary heritage of all livingEvidence of common evolutionary heritage of all livingthings (except human)things (except human)

Genes transcribed from one organism can be translated inGenes transcribed from one organism can be translated in

another organismanother organism Genes can be transferred from one organism to anotherGenes can be transferred from one organism to another

and be transcribed and translated in new hostand be transcribed and translated in new host

Second letter

U C A G


67/102

U C A G

Firstlet

ter(5end)

U

C

A

G

Third

letter(3

end)

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

= Stop Codon

= Start Codon


68/102

10.4.410.4.4TranscriptionTranscription

Transcribe only 1 of the 2 DNA strands :Transcribe only 1 of the 2 DNA strands : templatetemplatestrandstrand(antisense/- strand)(antisense/- strand)

DNA strand not transcribed : coding strand (sense/DNA strand not transcribed : coding strand (sense/

+ strand)+ strand) Synthesis in the 53 directionSynthesis in the 53 direction

No primer neededNo primer needed Bacteria 1 RNA polymeraseBacteria 1 RNA polymerase

Eukaryotes :Eukaryotes : RNA polymerase I synthesizes rRNARNA polymerase I synthesizes rRNA RNA polymerase II synthesizes mRNARNA polymerase II synthesizes mRNA RNA polymerase III synthesizes tRNARNA polymerase III synthesizes tRNA

Growing RNA

strand

Template

DNA strand


69/102

strand DNA strand

5' end 3' direction

Nucleotide

added to

growing

chain by

RNA polymerase

3' end 5' end

PromoterPromoter Promoter = binding site on template strand - start ofPromoter = binding site on template strand - start of


70/102

Promoter binding site on template strand start ofPromoter binding site on template strand start oftranscriptiontranscription

Short sequence of bases - not transcribed during transcriptionShort sequence of bases - not transcribed during transcription

Bacteria:Bacteria:

TTGACA (-35 sequence) located 35 nucleotides upstreamTTGACA (-35 sequence) located 35 nucleotides upstream(towards 5 end) of start site(towards 5 end) of start site

TATAATTATAAT (-10(-10 sequence) sequence) 1010 nucleotides upstreamnucleotides upstream

Eukaryotes:Eukaryotes:

TATA boxTATA box: similar to -: similar to -1010 sequence but located 25 nucleotidessequence but located 25 nucleotidesupstreamupstream

Promoter


71/102

Promoter


72/102

Stages in TranscriptionStages in Transcription

INITIATIONINITIATION

Transcription complexTranscription complexbinds tobinds topromoter, enabling binding of RNApromoter, enabling binding of RNApolymerasepolymerase

RNA polymerase unwinds DNA helixRNA polymerase unwinds DNA helixforming transcription bubbleforming transcription bubble

TRANSCRIPTION DNAEukaryotic promoters

1


73/102

Figure 17.8

RNA PROCESSING

TRANSLATION

Pre-mRNA

mRNA

Ribosome

Polypeptide

T A T A AA A

A T A T T T T

TATA box Start point Template

DNA strand

5

33

5

Transcription

factors

5

3

3

5

Promoter

5

3

3

55

RNA polymerase IITranscription factors

RNA transcript

Transcription initiation complex

Several transcription

factors

2

Additional transcription

factors

3


74/102

ELONGATIONELONGATION

RNA polymeraseRNA polymerase moves alongmoves alongtemplate strand - add ribonucleotidestemplate strand - add ribonucleotidesto 3 end of RNA strandto 3 end of RNA strand

The first 12 bases of new RNA strandThe first 12 bases of new RNA strand

temporarily forms helix with thetemporarily forms helix with thetemplate strandtemplate strand

ELONGATION (cont.)ELONGATION (cont.)


75/102

This stabilizes positioning of the 3 end of RNAThis stabilizes positioning of the 3 end of RNAso that it can add an incoming ribonucleotideso that it can add an incoming ribonucleotide

The RNA-DNA hybrid rotates each time aThe RNA-DNA hybrid rotates each time aribonucleotide is added to 3 endribonucleotide is added to 3 end

As RNA elongates, the earlier formed RNAAs RNA elongates, the earlier formed RNAstrand separates from template strandstrand separates from template strand

Template strand rewinds and reforms theTemplate strand rewinds and reforms thedouble helixdouble helix

ElongationNon-template


76/102

Non-template

strand of DNA

RNA

polymerase

RNA nucleotides

3 end3

5

5

Newly made

RNA

Template

strand of DNA

Direction of transcription(downstream)

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.


77/102

Templatestrand

Rewinding

mRNA

RNA-DNA hybrid helixRNA polymerase

Unwinding

Codingstrand

DNA

59

5'

39

39 59

39

C CC

C

C

C

C

C

C CCG

G GG

G GG G

G

GGAA

A AA

AA

A

A

A A

A

AT

T T

T

T

T

T

T

T T

T

T

U U

TERMINATIONTERMINATION


78/102

RNA polymerase reaches stop signalRNA polymerase reaches stop signal

RNA transcript at stop region forms aRNA transcript at stop region forms a CG hairpinCG hairpin

structure followed by 4 or more uracil (U)structure followed by 4 or more uracil (U)ribonucleotidesribonucleotides

Us allows RNA polymerase to release DNA templateUs allows RNA polymerase to release DNA template

RNA-DNA hybrid within transcription bubbleRNA-DNA hybrid within transcription bubbleseparatesseparates

DNA rewinds & transcription stopsDNA rewinds & transcription stops

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

C


79/102

C A

GC

CG

CG

GC

CG

U U U OH 3959

G

UG

UC

G

C

U

Promoter Transcription unit


80/102

35 DNAInitiation

RNA polymerase

Start point

Template strand

of DNARNA

tran-

script

Unwound

DNAElongation

3

3

53

5

5

3 5

Rewound

DNA

5 3

35 35RNA

transcript Termination

355 3

Completed RNA transcript

P t i ti lPostranscriptional


81/102

PostranscriptionalPostranscriptional

ModificationModification

Before moving from nucleus to cytoplasm,Before moving from nucleus to cytoplasm,each end of pre-mRNA are modified:each end of pre-mRNA are modified:

5 caps:5 caps:ATP or GTP forms 5 end of RNA strandATP or GTP forms 5 end of RNA strand A 5-5 linkage is then formed with GTP,A 5-5 linkage is then formed with GTP,

forming aforming a 5 cap5 cap Protects 5 end from nucleases &Protects 5 end from nucleases &

phosphatasesphosphatases


82/102

A modified guanine nucleotide

added to the 5 end

50 to 250 adenine nucleotides

added to the 3 end

Protein-coding segment Polyadenylation signal

Poly-A tail3 UTRStop codonStart codon

5 Cap 5 UTRAAUAAA AAAAAA

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATIONRibosome

Polypeptide

G P P P

5 3

UTR: Untranslated region


83/102

3 poly-A tails:3 poly-A tails: At the 3 end Poly-A polymeraseAt the 3 end Poly-A polymerase

enzyme adds aboutenzyme adds about 250250 AAribonucleotides at 3 end, formingribonucleotides at 3 end, forming 33poly-A tailpoly-A tail

Protects from nucleasesProtects from nucleases


84/102

10.4.5 RNA splicing10.4.5 RNA splicing

Eukaryotic gene is made up ofEukaryotic gene is made up of ExonsExons coding segments coding segments

IntronsIntrons non-coding segments non-coding segments Transcription producesTranscription producesprimary RNAprimary RNA

transcripttranscriptof entire geneof entire gene


85/102

Enzyme-RNA complex called small nuclearEnzyme-RNA complex called small nuclear


86/102

Enzyme-RNA complex called small nuclearEnzyme RNA complex called small nuclearribonucleoproteins (ribonucleoproteins (snRNPssnRNPs) recognize short) recognize shortnucleotide sequences at end of Intronsnucleotide sequences at end of Introns

Several different snRNPs associate with proteins toSeveral different snRNPs associate with proteins toformform splicesomesplicesome

Within splicesome, introns become folded intoWithin splicesome, introns become folded intoloops, bringing the exons close togetherloops, bringing the exons close together

Splicesome cuts Introns and joins together theSplicesome cuts Introns and joins together the

exons, forming shorterexons, forming shorter mature mRNA transcriptmature mRNA transcript

Exon IntronPromoter


87/102

DNA

Primary

RNAtranscript

MaturemRNAtranscript

5 cap 3 poly-A tailTRANSCRIPTION

RNA SPLICING


88/102

Promoter

Template

DNA strand

7-methylguanosine cap

1st

exon

1st

intron

2nd

exon

2nd

intron

3rd

exon

mRNA termination

sequence

Transcription, capping of 5 end

Stop codonStart codon

5 end


89/102


90/102


91/102


92/102


93/102

Process of TranslationProcess of Translation

a)a) INITIATIONINITIATION The small ribosomal subunit binds to the mRNA at the AUGThe small ribosomal subunit binds to the mRNA at the AUG

start codon.start codon.

The initiator tRNA or Methionine-tRNA (tRNAMet) withThe initiator tRNA or Methionine-tRNA (tRNAMet) with

anticodon UAC binds to the start codon (AUG codon) onanticodon UAC binds to the start codon (AUG codon) on

mRNA, and one of the initiation factors is released.mRNA, and one of the initiation factors is released. The large ribosomal subunit binds to the small subunit, andThe large ribosomal subunit binds to the small subunit, and

the remaining initiation factors are released.the remaining initiation factors are released.

TheThe initiation complexinitiation complexis complete.is complete.

Methionine subunit at P site; A & E sites are empty.Methionine subunit at P site; A & E sites are empty.


94/102


95/102

b)b) ELONGATIONELONGATION

2nd tRNA with complementary anticodon and2nd tRNA with complementary anticodon and

attached amino acid binds to A site with help ofattached amino acid binds to A site with help ofelongation factorselongation factors

Peptidyl transferasePeptidyl transferase breaks bond holding 1stbreaks bond holding 1stamino acid to 1st tRNA, and attaches it to 2ndamino acid to 1st tRNA, and attaches it to 2ndamino acid by peptide bondamino acid by peptide bond

1st tRNA is empty; 2nd tRNA has 2 amino acids1st tRNA is empty; 2nd tRNA has 2 amino acids

TRANSLOCATIONTRANSLOCATION Ribosome moves one codon in 53 direction with help ofRibosome moves one codon in 53 direction with help of

elongation factorselongation factors


96/102

elongation factorselongation factors

1st tRNA is shifted to E site; 2nd tRNA shifted to P site; A1st tRNA is shifted to E site; 2nd tRNA shifted to P site; Asite is emptysite is empty

3rd tRNA binds to A site3rd tRNA binds to A site Peptidyl transferase breaks bond holding 2nd amino acidPeptidyl transferase breaks bond holding 2nd amino acid

to 2nd tRNA, and attaches it to 3rd amino acidto 2nd tRNA, and attaches it to 3rd amino acid

2nd tRNA is empty; 3rd tRNA has 3 amino acids2nd tRNA is empty; 3rd tRNA has 3 amino acids

1st tRNA leaves the ribosome1st tRNA leaves the ribosome

Ribosome moves one codon, and whole process is repeatedRibosome moves one codon, and whole process is repeated


97/102


98/102

c)c) TERMINATIONTERMINATION

Ribosome reaches chain-terminatingRibosome reaches chain-terminating(stop) codon(stop) codon

Release factorsRelease factors signal ribosome tosignal ribosome torelease newly made polypeptiderelease newly made polypeptide

mRNA also releasedmRNA also released

Large & small subunits separateLarge & small subunits separate


99/102


100/102

Polypeptide

tRNA with

amino acid

attached

Ribosome

tRNA

Anticodon

35mRNA

Amino

acids

Codons


101/102

3

The release factor hydrolyzes the

bond between the tRNA in the

P site and the last amino acid of thepolypeptide chain. The polypeptide

is thus freed from the ribosome.

The two ribosomal subunits

and the other components

of the assembly dissociate.

Release

factor

Stop codon

(UAG, UAA, or UGA)

53

53

5

Free

polypeptide

When a ribosome reaches a stop

codon on mRNA, the A site of the

ribosome accepts a protein calleda release factor instead of tRNA.

10.4.7Differences Between Bacterial and10.4.7Differences Between Bacterial and


102/102

Eukaryotic Protein SynthesisEukaryotic Protein SynthesisEUKARYOTE BACTERIA

1. Most genes have introns 1. Most genes lack introns2. mRNA rarelyhave transcripts

of more than 1 gene2. mRNA often have transcripts

of several genes3. Translation begins after transcription is complete 3. Translation often beginsbefore transcription is

complete4. mRNA undergoes post-

transcriptional modification 5cap & 3 poly-A tail added

4. mRNA does not undergo post-transcriptional modification

5. mRNA modified before 5. mRNA not modified before

biology-dna & protein synthesis chapter 10

Documents