Chapter 9mgkmicro.com/BIOL257/Lecture10.pdf · Chapter 9 Topics - Genetics ... •pentose sugar...

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Chapter 9

Topics- Genetics- Flow of Genetics- Regulation- Mutation- Recombination

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Genetics

• Genome– Chromosome– Gene– Protein

• Genotype• Phenotype

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Termsandconcepts• gene– Fundamentalunitofheredity– DNAsegmentwhichcodesforproteinorRNA

• clone– populationofcellsthataregeneticallyidentical

• genome– allgenespresentinacellorvirus• haploid–onesetofgenes(eg.,bacteria)• diploid–twosetsofgenes(eg.,humans)

• genotype– specificsetofgenesanorganismpossesses

• phenotype– setofobservablecharacteristics

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The sum total of genetic material of a cell is referred to as thegenome.

Fig. 9.2 The general location and forms of the genome

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DNA and Chromosomes

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TheOrganizationofDNAinCells• Chromosomes–neatlypackagedDNAmolecule.

• organizationdiffersinprokaryoticandeukaryoticcelltypes

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Chromosome

• Prokaryotic– Histonelike proteins condense DNA

• Eukaryotic– Histone proteins condense DNA

• Subdivided into basic “informationalpackets” called genes

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Plasmids• usuallysmall,closedcircularDNAmolecules• existandreplicateindependentlyofchromosome• notrequiredforgrowthandreproduction• maycarrygenesthatconferselectiveadvantage(e.g.,drugresistance)

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Genes

• Three categories– Structural– Regulatory– Encode for functional/regulatory RNAs

• Genotype– Specific set of genes an organism possesses

• Phenotype– Set of observable characteristics

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Flow of Genetics

• NA replication (DNA => DNA; RNA => RNA)

• Gene Expression (DNA =>RNA=>Protein)– Replication– Transcription– Translation– Post-translational modification

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TheCentralDogma

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Representation of the flow of genetic information.

Fig. 9.9 Summary of the flow of genetic information in cell.

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DNA

• Structure - √

• Replication - today

Fig. 9.3 An Escherichia coli cell disrupted to release its DNA molecule.

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Structure

• Nucleotide– Phosphate– Deoxyribose sugar– Nitrogenous base

• Double stranded helix– Antiparallel arrangement

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DNAasGeneticMaterial• establishedbyseveralcriticalexperiments– FredGriffith(1928)– OswaldT.Avery,C.M.MacLeod,andM.J.McCarty(1944)– AlfredD.HersheyandMarthaChase(1952)

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Griffith’s Experiment

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Griffith’s Experiment: Transforming principle

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"fortheirdiscoveriesconcerningthemolecularstructureofnucleicacidsanditssignificanceforinformationtransferinlivingmaterial"

MauriceHughFrederickWilkins

JamesDeweyWatson

FrancisHarryComptonCrick

Structure of DNA

X-ray analysis of DNA structure

Rosalind Franklin

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DNAStructure• nitrogenousbases– A,T,G,C

• pentosesugar– deoxyribose

• chainofnucleotideslinkedbyphosphodiesterbonds

• usuallyadoublehelix,composedoftwocomplementarystrands– basepairingrules• AwithT• GwithC

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NucleicAcidStructure

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Nitrogenous bases

• Purines– Adenine– Guanine

• Pyrimidines– Thymine– Cytosine

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DNAStructure

23two polynucleotide chains are anti-parallel

DNAStructure

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RNA Structure

• nitrogenous bases– A, G, C, U (instead of T)

• pentose sugar– ribose

• usually consists of single strand of nucleotides linkedby phosphodiester bonds– can coil back on itself

• forms hairpin-shaped structures with complementarybase pairing and helical organization

• base pairing rules– A with U– G with C

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Prokaryotic chromosome

• usually exists asclosed circular,supercoiledmoleculeassociated withbasic proteins

relaxed circle

supercoiled DNA

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EukaryoticDNA• linearmolecules• associatedwithhistones• coiledintorepeatingunitscallednucleosomes

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EukaryoticChromosomes

A chromosome is formed from asingle DNA molecule thatcontains many genes.

A chromosomal DNA moleculecontains three specificnucleotide sequences which arerequired for replication: a DNAreplication origin;a centromere to attach the DNAto the mitotic spindle.;a telomere located at each endof the linear chromosome.

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Purines and pyrimidines pair (A-T or G-C) and the sugars(backbone) are linked by a phosphate.

Fig. 9.4 Three views of DNA structure

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Replication

• Nature: Semiconservative• Involved Enzymes (in Primosome & Replisome)

• Leading strand• Lagging strand

– Okazaki fragments

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Semiconservative

• New strands are synthesized in 5’ to 3’direction

Minus (–)strandof DNA

3' 5'

T A C G A C T A A T A G G C G C A T C C A C G A T C

Plus (+)strandof DNA

5' 3'


3' 5'


A T G C T G A T T A T C C G C G T A G G T G C T A G

A T G C T G A T T A T C C G C G T A G G T G C T A G

A U G C U G A U U A U C C G C G U A G G U G C U A G

Plus (+)strandof DNA


5' 3'

3' 5'

5' 3' mRNA


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Semiconservative replication of DNA synthesizes a new strand of DNA from atemplate strand.

Fig. 9.5 Simplified steps to show the semiconservative replication of DNA

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Enzymes

• Helicase• DNA polymerase III• Primase• DNA polymerase I• Ligase• Gyrase

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The function of important enzymes involved in DNA replication.

Table 9.1 Some enzymes involved in DNA replication

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Leading strand

• DnaA,DnaB proteins at Origin or Replication(circular genetic element) or

• RNA primer (linear genetic element) initiate the 5’ to3’ synthesis of DNA in a continuous manner

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Lagging strand

• Because the direction of synthesis (5’ -> 3’) isopposite to fork movement, the lagging strand issynthesized in form of multiple DNA (Okazaki)fragments

• Primer synthesis performed by RNA polymerase (Primase),• DNA synthesis performed by DNA polymerase III• RNA primer removal and fill-in with DNA by DNA polymerase I

• Okazaki fragments are ligated together by DNAligase to form one continuous strand

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The steps associated with the DNA replication process.

Fig. 9.6 Thebacterialreplicon: amodel for DNASynthesis

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PatternsofDNAsynthesis…• inprocaryotes– bidirectionalfromasingleoriginofreplication– replicon• portionofthegenomethatcontainsanoriginandisreplicatedasaunit

2 forks movein oppositedirections

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MechanismofDNAReplication in bacteria

unwind strands and relievetension caused by unwinding

keep strands separated

synthesizedby primase

DNA polymerase III synthesizes DNA

Quinolone Target

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Someamazingfacts• ≥30proteinsrequiredtoreplicateE.colichromosome• occurswithgreatfidelity– errorfrequency=10-9or10-10perbasepairreplicated– duetoproofreadingactivityofDNApolymerasesIIIandI

• occursveryrapidly– 750to1,000basepairs/secondinprocaryotes– 50-100basepairs/secondineucaryotes

5'3'

Helicase separatesthe double-strandedmolecule

5'3'

Synthesis of the leadingstrand proceeds continuously.

3'5'


5'3'


Synthesis of the lagging strand isdiscontinuous; synthesis is reinitiatedperiodically, generating a series offragments that are later joined.

1. Primase synthesizes an RNA primer

3'5'


RNA primer

5'3'




3'5'


2. DNA polymerase adds nucleotides onto the 3' end of the fragment

RNA primer

5'3'

3'5'




3. DNA polymerase replaces the RNA primer with deoxynucleotides



RNA primer

5'3'

3'5'




3. DNA polymerase replaces the RNA primer with deoxynucleotides

3'5'



RNA primer

4. DNA ligase seals the gaps between adjacent fragments

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Replication processes from other biological systems (some Gram-positive bacterial genomes, plasmids, viruses) involve a rolling cycle.

Fig. 9.8 Simplified model of rolling circle DNA Replication

5’3’

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RNA

• Transcription– Message RNA (mRNA)– Transfer RNA (tRNA)– Ribosomal RNA (rRNA)

• Codons (nucleotide triplett)

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(a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.*

*For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA.

3′DNA SampleDNA SampleCycle 1

Heat to 94°C3′5′

5′

Polymerase Chain Reaction

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Heat to 94°CDenaturationDenaturation

Cycle1

Strandsseparate

3′

3′

3′ 5′

5′5′

5′

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Heat to 94°C

50° to 65°C

DenaturationDenaturation

Oligonucleotideprimers attach atends of strands topromote replicationof amplicons

PrimerPrimer Cycle1

Strandsseparate

Amplicons

PrimingPriming

3′

3′

3′

3′

3′

3′ 5′

5′

5′5′

5′

5′

5′3′ 5′

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Heat to 94°C

50° to 65°C



PrimerPrimer

ExtensionExtensionDNA polymerasesynthesizescomplementary strand

Cycle1

Strandsseparate

72°C

Amplicons

PrimingPriming

3′

3′

3′

3′3′

3′

3′

3′

3′

3′

5′

5′

5′

5′5′

5′

5′

5′

5′3′ 5′

5′

Polymerase5′

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DenaturationDenaturation New strandOriginalstrands

Heatto

94°C

Cycle2

New strand

Cycle 22 copies

Heat to 94°C

50° to 65°C



PrimerPrimer


Cycle1

Strandsseparate

72°C

Amplicons

PrimingPriming

3′

3′

3′

3′3′

3′

3′

3′

3′

3′

5′

5′

5′

5′5′

5′

5′

5′

5′3′ 5′

5′

Polymerase5′

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PrimingPriming

New strandOriginalstrands

Heatto

94°C

Cycle2

New strand

Cycle 22 copies

72°C

Heat to 94°C

50° to 65°C



PrimerPrimer


Cycle1

Strandsseparate

72°C

Amplicons

PrimingPriming

3′

3′

3′

3′3′

3′

3′

3′

3′

3′

5′

5′

5′

5′5′

5′

5′

5′

5′3′ 5′

5′

Polymerase5′

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PrimingPriming


Heatto

94°C

Cycle2

New strand

ExtensionExtension

Cycle 22 copies

4 copies

72°C

Heat to 94°C

50° to 65°C



PrimerPrimer


Cycle1

Strandsseparate

72°C

Amplicons

PrimingPriming

3′

3′

3′

3′3′

3′

3′

3′

3′

3′

5′

5′

5′

5′5′

5′

5′

5′

5′3′ 5′

5′

Polymerase5′

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(b) A view of the process after 6 cycles, with 64 copies of amplified DNA. Continuing this process for 20 to 40 cycles can produce millions of identical DNA molecules.




PrimingPriming


Heatto

94°C

Cycle2

Cycles 3, 4, . . . repeat same steps

New strand

ExtensionExtension

Cycle 22 copies

4 copies

72°C

Heat to 94°C

50° to 65°C



PrimerPrimer


Cycle1

Strandsseparate

72°C

Amplicons

PrimingPriming

1* fragment2 copies

4 copies8 copies

16 copies32 copies64 copies

3′

3′

3′

3′3′

3′

3′

3′

3′

3′

5′

5′

5′

5′5′

5′

5′

5′

5′3′ 5′

5′

Polymerase5′

Chapter 9mgkmicro.com/BIOL257/Lecture10.pdf · Chapter 9 Topics - Genetics ... •pentose sugar...

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