Chapter 10 Molecular Biology of the Gene

Chapter 10Chapter 10

Molecular Biology of the GeneMolecular Biology of the Gene

Molecular Biology: Molecular Biology:

Study of DNA and how it serves as the molecular Study of DNA and how it serves as the molecular basis of heredity.basis of heredity.

In 1920s it became clear that chromosomes In 1920s it became clear that chromosomes contained genes for genetic traits. contained genes for genetic traits.

However, chromosomes are made up of both However, chromosomes are made up of both protein and DNA. protein and DNA.

Which one was the genetic material?Which one was the genetic material?

Before the 1940s most scientists believed that Before the 1940s most scientists believed that proteins were the genetic material of cells, and proteins were the genetic material of cells, and that nucleic acids (DNA and RNA) were too that nucleic acids (DNA and RNA) were too simple to code for genes.simple to code for genes.

Molecular Biology: Molecular Biology:

How was the genetic material identified?How was the genetic material identified?

A number of experiments were important in A number of experiments were important in

establishing that DNA was indeed the genetic establishing that DNA was indeed the genetic

material of living organisms.material of living organisms. Frederick Griffith’s experiments (1928)Frederick Griffith’s experiments (1928)

Oswald Avery’s experiments (1944)Oswald Avery’s experiments (1944)

Alfred Hershey and Martha Chase experiments (1954)Alfred Hershey and Martha Chase experiments (1954)

I. I. Frederick Griffith’s ExperimentsFrederick Griffith’s Experiments (1928) (1928)

Griffith was studying two strains of the bacteria Griffith was studying two strains of the bacteria Streptococcus pneumoniaeStreptococcus pneumoniae, , which causes which causes pneumonia and other infections.pneumonia and other infections.

Smooth strainSmooth strain (S): (S): Produced a polysaccharideProduced a polysaccharide capsulecapsule

that gave its colonies a smooth appearance. that gave its colonies a smooth appearance.

Because of the capsule, the bacteria can evade the Because of the capsule, the bacteria can evade the

immune system and immune system and causecause diseasedisease..

Rough strainRough strain (R): (R): Does not produce a capsule, rough Does not produce a capsule, rough

appearance. appearance.

Not pathogenic, Not pathogenic, doesn’tdoesn’t causecause diseasedisease..


Griffith treated his mice with the following Griffith treated his mice with the following bacterial preparations:bacterial preparations:

TreatmentTreatment OutcomeOutcome

1. Live rough1. Live rough Mouse lives Mouse lives

2. Live smooth2. Live smooth Mouse dies Mouse dies

3. Heat killed smooth3. Heat killed smooth Mouse lives Mouse lives

4. Heat killed smooth + live rough4. Heat killed smooth + live rough Mouse dies Mouse dies

To his surprise, he did not get the expected To his surprise, he did not get the expected results in the last treatment (#4). The mice got results in the last treatment (#4). The mice got sick and died. Additionally, he recovered sick and died. Additionally, he recovered smooth bacteria from these mice. smooth bacteria from these mice.

Griffith’s Experiments: Transformation of HarmlessBacteria into Deadly Bacteria


Why were smooth bacteria present in the last Why were smooth bacteria present in the last

group of mice (live rough + dead smooth)?group of mice (live rough + dead smooth)?

Griffith concluded that the Griffith concluded that the genetic instructionsgenetic instructions to to

make capsules had been make capsules had been transferredtransferred to the rough to the rough

bacteria, from the dead smooth bacteria.bacteria, from the dead smooth bacteria.

He called this phenomenon He called this phenomenon transformationtransformation..

However, he was unable to identify what type of However, he was unable to identify what type of

molecule was responsible for transformation.molecule was responsible for transformation.

II. II. Oswald Avery’s ExperimentsOswald Avery’s Experiments (1944) (1944)

Avery repeated Griffith’s experiments using Avery repeated Griffith’s experiments using purified DNA, protein, and other substances.purified DNA, protein, and other substances.

He showed that the chemical substance He showed that the chemical substance responsible for responsible for transformationtransformation was was DNADNA and and not not protein.protein.

While many biologists were convinced, others While many biologists were convinced, others remained skeptical.remained skeptical.

III. III. Hershey and Chase ExperimentsHershey and Chase Experiments (1952): (1952): Definitive proof that DNA rather than Protein Definitive proof that DNA rather than Protein carries the hereditary information of lifecarries the hereditary information of life

E. Coli E. Coli bacteriophagebacteriophage: : A virus that infects bacteria.A virus that infects bacteria.

Bacteriophages only contain a protein coat Bacteriophages only contain a protein coat ((capsidcapsid) and DNA. ) and DNA.

They wanted to find out whether the They wanted to find out whether the proteinprotein or or DNADNA carried the genetic instructions to make carried the genetic instructions to make more viruses.more viruses.

They labeled either the viral proteins or DNA:They labeled either the viral proteins or DNA: Protein capsid:Protein capsid: Labeled with radioactive sulfur (Labeled with radioactive sulfur (3535S)S) DNA:DNA: Labeled with radioactive phosphorus (Labeled with radioactive phosphorus (3232P)P)

Radioactive labeled viruses were used to infect Radioactive labeled viruses were used to infect cells.cells.

Bacteriophages Are Viruses that Infect Bacteria

Either Bacteriophage DNA or Proteins Can be Labeled with Radioactive Elements

Hershey Chase Experiment: DNA is Genetic Material

III. III. Hershey and Chase ExperimentsHershey and Chase Experiments (1952): (1952):

Bacterial cells that were infected with the two Bacterial cells that were infected with the two types of bacteriophage, were then spun down types of bacteriophage, were then spun down into a pellet (centrifuged), and examined.into a pellet (centrifuged), and examined.

ResultsResults::1. Labeled viral 1. Labeled viral proteins did not enterproteins did not enter infected bacteria infected bacteria

(found in supernatant).(found in supernatant).

2. Labeled viral 2. Labeled viral DNA did enterDNA did enter bacteria during viral bacteria during viral

infection (found in cell pellet)infection (found in cell pellet)..

ConclusionConclusion::

Protein is not necessary to make new viruses.Protein is not necessary to make new viruses.

DNA is the molecule that carries the genetic DNA is the molecule that carries the genetic

information to make new viruses!!!!information to make new viruses!!!!

IV.IV. DNA and RNA are Polymers of Nucleotides DNA and RNA are Polymers of Nucleotides

Each nucleotide has:Each nucleotide has:

1. A 5 carbon sugar (pentose):1. A 5 carbon sugar (pentose): Deoxyribose in DNADeoxyribose in DNA Ribose in RNARibose in RNA

2. A phosphate group.2. A phosphate group.

3. A N-containing organic base:3. A N-containing organic base: DNADNA: Adenine (A), Guanine (G), Cytosine (C), and : Adenine (A), Guanine (G), Cytosine (C), and

Thymine (T).Thymine (T). RNARNA:: Adenine (A), Guanine (G), Cytosine (C), and Adenine (A), Guanine (G), Cytosine (C), and

Uracil (U).Uracil (U). PurinesPurines: Bases with two rings (A and G): Bases with two rings (A and G) PyrimidimesPyrimidimes: Bases with one ring (C, T, and U): Bases with one ring (C, T, and U)

DNA are Polymers of Nucleotides

IV. Structure of DNA MoleculeIV. Structure of DNA Molecule

Erwin ChargaffErwin Chargaff (1947) (1947)

Chargaff’s Rule: In the DNA of all living Chargaff’s Rule: In the DNA of all living organisms, the amount of A = T and the G = Corganisms, the amount of A = T and the G = C

No matter which species on earth he studiedNo matter which species on earth he studied, the , the DNA showed the same relative ratiosDNA showed the same relative ratios Adenine = ThymineAdenine = Thymine Guanine = CytosineGuanine = Cytosine

These results suggested that A & T and C & G These results suggested that A & T and C & G were somehow were somehow paired uppaired up with each other in a with each other in a DNA molecule.DNA molecule.

IV. IV. Rosalind Franklin, James Watson, and Francis Rosalind Franklin, James Watson, and Francis CrickCrick (April 1953, (April 1953, NatureNature)) Rosalind FranklinRosalind Franklin: : X-ray crystallography of DNA, X-ray crystallography of DNA,

trying to determine the structure of the molecule. trying to determine the structure of the molecule. Franklin’s work laid the foundation for Watson and Franklin’s work laid the foundation for Watson and Crick. Died of cancer in 1958.Crick. Died of cancer in 1958.

James Watson and Francis CrickJames Watson and Francis Crick: Determined the : Determined the exact three dimensional structure of DNA as a double exact three dimensional structure of DNA as a double helix helix held together byheld together by H bondsH bonds. . Won 1962 Nobel Prize.Won 1962 Nobel Prize.

DNA is an DNA is an antiparallelantiparallel double helix: 5’ end of one double helix: 5’ end of one strand is paired to 3’ end of other strand.strand is paired to 3’ end of other strand. A & T and G & C are paired up by hydrogen bondsA & T and G & C are paired up by hydrogen bonds Two strands are Two strands are complementarycomplementary to each other. to each other. If you know sequence of one strand, can determine If you know sequence of one strand, can determine

sequence of the other one.sequence of the other one.

DNA Structure: Double Helix Held Together by H Bonds

Hydrogen Bonding Between Complementary Nucleotides Explains Chargaff’s Rule

V. How exactly does DNA replicate?V. How exactly does DNA replicate?

Several models for DNA replication were proposed: Several models for DNA replication were proposed:

1. 1. Conservative modelConservative model:: Two completely new Two completely new

strands are formed, which coil together. strands are formed, which coil together.

Original strands stay together.Original strands stay together.

2. 2. Semiconservative modelSemiconservative model: One original strand : One original strand

pairs up with one new strand.pairs up with one new strand.

3. 3. Dispersive modelDispersive model: Each strand is a mixture of : Each strand is a mixture of

old and new DNA.old and new DNA.

Three Models of DNA Replication

V. How exactly does DNA replicate?V. How exactly does DNA replicate?

FindingsFindings:: Replication is carried out by Replication is carried out by DNA polymeraseDNA polymerase..

50 nucleotides per second in mammals50 nucleotides per second in mammals 500 nucleotides per second in bacteria 500 nucleotides per second in bacteria

DNA strands unzipDNA strands unzip and each one acts as a template for and each one acts as a template for the formation of a new strand.the formation of a new strand.

Nucleotides line up along template strand in Nucleotides line up along template strand in accordance with accordance with base pairing rulesbase pairing rules..

Enzymes link the nucleotides together to form new Enzymes link the nucleotides together to form new DNA strands.DNA strands.

SemiconservativeSemiconservative replicationreplication: Each new helix will : Each new helix will contain one new strand and one old strand.contain one new strand and one old strand.

DNA Replication is Semiconservative

V. How exactly does DNA replicate?V. How exactly does DNA replicate? Strands are antiparallelStrands are antiparallel: : Run in opposite Run in opposite

directions.directions.

DNA polymerasesDNA polymerases can only add nucleotides to can only add nucleotides to

one end of the strand (3’ end).one end of the strand (3’ end).

New strands grow in a New strands grow in a 5’ to 3’ direction5’ to 3’ direction

Replication forkReplication fork with: with:

Leading strandLeading strand: Made continuously.: Made continuously.

Lagging strandLagging strand: Made discontinuously in : Made discontinuously in

Okazaki fragmentsOkazaki fragments which are then joined which are then joined

together.together.

DNA Strands are Antiparallel

DNA Replication: Double Helix Must UnwindTwo Strands are Made Differently

DNA Replication: Leading Strand is Made Continuously;Lagging Strand is Made in Fragments

VI. DNA Genotype is Expressed VI. DNA Genotype is Expressed Phenotypically as ProteinPhenotypically as Protein GeneGene: Segment of DNA that codes for a protein : Segment of DNA that codes for a protein

or RNA product. or RNA product.

Fundamental unit of heredity. Fundamental unit of heredity.

DNA sequences specify order of amino acids in DNA sequences specify order of amino acids in protein; but do not produce protein directly.protein; but do not produce protein directly.

Proteins are crucial to cell activityProteins are crucial to cell activity Cell movementCell movement Oxygen and carbon dioxide transportOxygen and carbon dioxide transport Active transport across membranesActive transport across membranes Cell divisionCell division Enzymatic reactions (respiration, digestion, etc.)Enzymatic reactions (respiration, digestion, etc.)

VII. Flow of Genetic Information in the CellVII. Flow of Genetic Information in the Cell DNA does not produce protein directly. DNA does not produce protein directly.

Genetic blueprints do not leave nucleus.Genetic blueprints do not leave nucleus. TranscriptionTranscription: An RNA copy of gene is made : An RNA copy of gene is made

((messenger RNAmessenger RNA or or mRNAmRNA). ). In In eukaryoteseukaryotes mRNA is made in nucleus and then goes mRNA is made in nucleus and then goes

to cytoplasm.to cytoplasm.

TranslationTranslation: Process in which protein is made : Process in which protein is made from instructions contained in from instructions contained in messenger RNAmessenger RNA. .

Translation is carried out by Translation is carried out by ribosomesribosomes in the in the cytoplasm or rough E.R.cytoplasm or rough E.R.

VII. Flow of Genetic Information in the Cell VII. Flow of Genetic Information in the Cell

TranscriptionTranscription TranslationTranslation

DNA DNA mRNA mRNA Protein Protein

Transcription and Translation Occur in Different Parts of Eucaryotic Cells

VIII. RNA is made by TranscriptionVIII. RNA is made by Transcription Occurs in the cell’s nucleus.Occurs in the cell’s nucleus. TranscriptionTranscription is carried out by is carried out by RNA polymerase.RNA polymerase. A single strand of DNA (“A single strand of DNA (“coding strandcoding strand”) serves ”) serves

as the template for RNA synthesis.as the template for RNA synthesis. RNA nucleotides are matched to complementary RNA nucleotides are matched to complementary

DNA nucleotides in 5’ to 3’ direction.DNA nucleotides in 5’ to 3’ direction. RNA contains RNA contains uracil (U)uracil (U) instead of thymine (T). instead of thymine (T). mRNA transcript leaves nucleus through nuclear mRNA transcript leaves nucleus through nuclear

pores and goes to cytoplasm. pores and goes to cytoplasm.

Transcription of a Gene

IX. Proteins are made by TranslationIX. Proteins are made by Translation Occurs on ribosomes in the cell’s cytoplasm or Occurs on ribosomes in the cell’s cytoplasm or

rough ER.rough ER. TranslationTranslation is a complex process, carried out by is a complex process, carried out by

several types of RNA molecules:several types of RNA molecules:

1. Messenger RNA (mRNA)1. Messenger RNA (mRNA)

2. Transfer RNA (tRNA)2. Transfer RNA (tRNA)

3. Ribosomal RNA (rRNA)3. Ribosomal RNA (rRNA)

Necessary Components for Translation:Necessary Components for Translation:

1. Messenger RNA (mRNA):1. Messenger RNA (mRNA): Encodes for a Encodes for a

specific protein sequence.specific protein sequence.

Variable lengthVariable length (depending on protein size) (depending on protein size)

Information is read in Information is read in tripletstriplets ( (codonscodons))

64 possible codons (4 x 4 x 4 = 64 = 464 possible codons (4 x 4 x 4 = 64 = 433))

• 61 codons specify amino acids61 codons specify amino acids

• 3 codons are termination signals.3 codons are termination signals.

mRNA is complementary to DNA and read in triplets (codons)


2. Transfer RNA (tRNA):2. Transfer RNA (tRNA): Brings one amino Brings one amino acid at a time to the growing polypeptide acid at a time to the growing polypeptide

chainchain.. SmallSmall molecule (70 to 90 nucleotides)molecule (70 to 90 nucleotides)

Forms a cloverlForms a cloverleaf structureeaf structure AnticodonAnticodon: Base pairs to mRNA codon during : Base pairs to mRNA codon during

translation.translation. Amino acid binding siteAmino acid binding site: At 3’ end of : At 3’ end of

molecule.molecule.

Transfer RNA (tRNA) Carries Amino Acids to the Growing Polypeptide Chain


3. Ribosomal RNA (rRNA):3. Ribosomal RNA (rRNA): Ribosome is site of Ribosome is site of

protein synthesisprotein synthesis..

Facilitates coupling of mRNA to tRNAFacilitates coupling of mRNA to tRNA

Huge moleculeHuge molecule: Large and small subunits : Large and small subunits

must assemble for translation.must assemble for translation.

Ribosome compositionRibosome composition: 60% rRNA and 40% : 60% rRNA and 40%

proteinprotein

Ribosome is the Site of Translation

STEPS OF TRANSLATION:STEPS OF TRANSLATION:

1. INITIATION:1. INITIATION: Messenger RNA (mRNA) and ribosome come together.Messenger RNA (mRNA) and ribosome come together.

Transfer RNA (tRNA):Transfer RNA (tRNA): Carrying first amino acid Carrying first amino acid

(methionine) has (methionine) has anticodonanticodon which binds to which binds to start codonstart codon

(AUG).(AUG).

2. ELONGATION2. ELONGATION One amino acid at time is added and linked to growing One amino acid at time is added and linked to growing

polypeptide chain by a peptide bond.polypeptide chain by a peptide bond.

3. TERMINATION3. TERMINATION Stop codonsStop codons: UAA, UAG, or UGA: UAA, UAG, or UGA

Ribosome/mRNA complex dissociates.Ribosome/mRNA complex dissociates.

Translation: Initiation at Start Codon

Translation: During Elongation one Amino Acid is Added at a Time

Elongation: Ribosome Travels Down mRNA, Adding One Amino Acid at a Time

Termination: Once Stop Codon is Reached, Complex Disassembles

X. Genetic CodeX. Genetic Code Twenty amino acids are found in proteinsTwenty amino acids are found in proteins

Four nucleotides are found in RNA and DNAFour nucleotides are found in RNA and DNA

How can nucleic acids with only 4 bases encode proteins How can nucleic acids with only 4 bases encode proteins

with 20 amino acids?with 20 amino acids?

Each amino acid is encoded by more than one nucleotideEach amino acid is encoded by more than one nucleotide If 1 base = 1 amino acid, Can only determine 4 amino acidsIf 1 base = 1 amino acid, Can only determine 4 amino acids

If 2 bases = 1 amino acid, Can only determine 16 amino acidsIf 2 bases = 1 amino acid, Can only determine 16 amino acids

If 3 bases (Codon)= 1 amino acid, Can determine 64 amino acids If 3 bases (Codon)= 1 amino acid, Can determine 64 amino acids

Genetic code is Genetic code is redundantredundant, more than one codon per , more than one codon per

several amino acids.several amino acids.

Genetic code is Genetic code is universaluniversal

Universal Genetic Code

Mutations are permanent changes in DNAMutations are permanent changes in DNA

DNA replication is never 100% accurateDNA replication is never 100% accurate

Bases may be inserted, deleted, or mismatched Bases may be inserted, deleted, or mismatched

during replication.during replication.

Mutations:Mutations: Any mistakes that cause changes in Any mistakes that cause changes in

the nucleotide sequence of DNA. the nucleotide sequence of DNA.

Mutations may be either harmful, beneficial, or Mutations may be either harmful, beneficial, or

have no effect on a cell or individual.have no effect on a cell or individual.

XI. Mutations: Permanent changes in DNAXI. Mutations: Permanent changes in DNA

There are several possible types of mutations:There are several possible types of mutations:

I. I. Substitution mutation:Substitution mutation: One nucleotide is One nucleotide is replacedreplaced by by

another. May result in:another. May result in:

Missense:Missense: Different amino acid. May or may not have Different amino acid. May or may not have

serious consequences. serious consequences.

Example: Sickle cell anemia.Example: Sickle cell anemia.

Nonsense:Nonsense: Stop codon. Protein is truncated. Usually Stop codon. Protein is truncated. Usually

has serious consequences.has serious consequences.

Silent:Silent: No change in amino acid. No consequence.No change in amino acid. No consequence.

Missense Mutation in Sickle Cell Anemia

Base substitution results in a single amino acid change Glu ---> Val

XI. Mutations: Permanent changes in DNAXI. Mutations: Permanent changes in DNA

There are several possible types of mutations:There are several possible types of mutations:

II. II. Frameshift Mutation:Frameshift Mutation: Nucleotides which are Nucleotides which are insertedinserted or or

deleteddeleted may may changechange the gene’s the gene’s reading framereading frame. .

Usually serious, because entire protein sequence after Usually serious, because entire protein sequence after

mutation may be disrupted.mutation may be disrupted.

Effects of Different Types of Mutations

Many Viruses Cause Disease in AnimalsMany Viruses Cause Disease in AnimalsReproductive Cycle of an Animal VirusReproductive Cycle of an Animal Virus

1. 1. Entry:Entry: Virus gets inside cell.Virus gets inside cell. Attachment:Attachment: Virus attaches to a specific receptor on cell Virus attaches to a specific receptor on cell

surface.surface. Penetration:Penetration: Virus fuses to cell membrane and enters cell.Virus fuses to cell membrane and enters cell.

2. 2. Uncoating:Uncoating: Viral capsid releases genetic material. Viral capsid releases genetic material.

3. 3. Synthesis:Synthesis: Genetic material is copied, viral proteins Genetic material is copied, viral proteins are made.are made.

4. 4. Assembly:Assembly: Genetic material is packaged into capsids. Genetic material is packaged into capsids. Release:Release: New viruses (50-200) leave the cell through:New viruses (50-200) leave the cell through:

Lysis:Lysis: Cells burst and die.Cells burst and die. Budding:Budding: Cell does not necessarily die.Cell does not necessarily die.

Life Cycle of the Influenza (Flu) Virus

The AIDS Virus Makes DNA from RNAThe AIDS Virus Makes DNA from RNA

Human Immunodeficiency Virus (HIV): Causes AIDS. Human Immunodeficiency Virus (HIV): Causes AIDS. HIV is a retrovirus, which contains the enzyme reverse HIV is a retrovirus, which contains the enzyme reverse transciptase.transciptase.

Flow of genetic information is reversed:Flow of genetic information is reversed:

TranscriptionTranscription TranslationTranslation

DNA DNA RNA RNA Protein Protein

ReverseTranscription

Viral DNA is inserted into host chromosomeas a provirus.

HIV Contains Unique Enzyme Reverse Transcriptase

Infection of a Cell by HIV

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Chapter 10 Molecular Biology of the Gene